Compositions and methods for treating progressive myocardial injury due to a vascular insufficiency

ABSTRACT

The described invention provides methods and regimens for treating adverse consequences of a persistent and progressive myocardial injury-due to a vascular insufficiency that occurs early or late in a subject in need thereof, and progressive myocardial injury-preventing compositions that contain a chemotactic hematopoietic stem cell product, and, optionally, an additional active agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/910,328 (filed Oct. 22, 2010), which claims the benefit of priorityto U.S. Provisional Application No. 61/254,539 (filed Oct. 23, 2009) andis a continuation-in-part of U.S. application Ser. No. 12/629,361 (filedDec. 2, 2009), which claims the benefit of priority to U.S. ProvisionalApplication Nos. U.S. 61/119,552 (filed Dec. 3, 2008) and U.S.61/169,850 (filed Apr. 16, 2009) and a continuation-in-part of U.S.11/552,396 (filed Oct. 24, 2006), which issued as. U.S. Pat. No.7,794,705, which claims the benefit of priority to U.S Provisionalapplication Ser. No. 60/734,151 (filed Nov. 7, 2005). U.S. applicationSer. No. 12/910,328 is also a continuation-in-part of U.S. applicationSer. No. 12/401,291 (filed Mar. 10, 2009), which issued as U.S. Pat. No.8.088,370 which is a division of of U.S. application Ser. No.11/552,396, which issued as U.S. Pat. No. 7,794,705. Each of theseapplications is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The described invention relates to compositions comprising a chemotactichematopoietic stem cell product and methods of use thereof for treatingearly or late adverse consequences of vascular insufficiency.

BACKGROUND OF THE INVENTION The Cardiac Cycle

The term “cardiac cycle” is used to refer to all or any of themechanical events related to the coronary blood flow or blood pressurethat occurs from the beginning of one heartbeat to the beginning of thenext. Blood pressure increases and decreases throughout the cardiaccycle. The frequency of the cardiac cycle is the heart rate. Everysingle ‘beat’ of the heart involves five major stages: (1)“latediastole,” which is when the semilunar valves close, theatrioventricular (Av) valves open and the whole heart is relaxed; (2)“atrial systole,” which is when the myocardium of the left and rightatria are contracting, AV valves open and blood flows from atrium toventricle; (3) “isovolumic ventricular contraction,” which is when theventricles begin to contract, AV and semilunar valves close, and thereis no change in volume; (4) “ventricular ejection,” which is when theventricles are empty but still contracting and the semilunar valves areopen; and (5) “isovolumic ventricular relaxation,” when pressuredecreases, no blood is entering the ventricles, the ventricles stopcontracting and begin to relax, and the semilunar valves are shutbecause blood in the aorta is pushing them shut. The cardiac cycle iscoordinated by a series of electrical impulses that are produced byspecialized heart cells found within the sino-atrial node and theatrioventricular node.

Coronary Blood Flow

The flow of blood through the coronary arteries is pulsatile, withcharacteristic phasic systolic and diastolic flow components. Systolicflow, which relates to the contraction or pumping phase of the heartcycle, has rapid, brief, retrograde responses. Diastolic flow, whichrelates to the relaxation or filling phase of the heart cycle, occursduring the relaxation phase after myocardial contraction, with an abruptincrease above systolic levels and a gradual decline parallel with thatof aortic diastolic pressures. Intramural coronary blood volume changesduring each heartbeat, with the myocardium accommodating the volumechange brought about by muscular contraction. Coronary venous flow isout of phase with coronary arterial flow, occurring predominantly insystole and nearly absent during diastole.

For each heartbeat, blood pressure varies between systolic and diastolicpressures. The term “systolic pressure” refers to the peak pressure inthe arteries, which occurs near the end of the cardiac cycle when theventricles are contracting. The term “diastolic pressure” refers to theminimum pressure in the arteries, which occurs near the beginning of thecardiac cycle when the ventricles are filled with blood.

Coronary blood flow not only is phasic but also varies with the type ofvessel and location in the myocardium. Coronary arterioles appear tohave specialized regulatory elements along their length that operate “inseries” in an integrated mariner. A system of multiple functional“valves” permits fine control of the coronary circulation. The smallestarterioles dilate during metabolic stress, resulting in reducedmicrovascular resistance and increased myocardial perfusion. Stenosis ornarrowing of a blood vessel produces resistance to blood flow relateddirectly to the morphologic features of the stenosis. As the upstreamarteriolar pressure decreases due to a fall in distending pressureacross the stenosis, myogenic dilation of slightly larger arteriolesupstream occurs and causes an additional decrease in resistance.Increased flow in the largest arterioles augments shear stress andtriggers flow-mediated dilation, further reducing the resistance of thisnetwork.

The arterial and venous pulsatile flow characteristics of the heart aredependent on intramyocardial compliance. The term “compliance” refers toa measure of the tendency of a hollow organ to resist recoil toward itsoriginal dimensions upon removal of a distending or compressing force.The higher the compliance the more elastic the material. Compliance iscalculated using the following equation, where ΔV is the change involume, and ΔP is the change in pressure:C=ΔV/ΔP

The capacity of the heart as a reservoir is controlled by resistancearterioles to coronary vascular inflow. Outlet resistance is related tointramural cardiac veins. The intramyocardial capillary resistanceinfluences both arterial and venous responses but predominantly acts inconcert with outlet resistance.

Approximately 75% of total coronary resistance occurs in the arterialsystem, which comprises conductance (R1), prearteriolar (R2) andarteriolar and intramyocardial capillary vessels (R3). Normal epicardialcoronary arteries in humans typically are 0.3 to 5 mm in diameter, anddo not offer appreciable resistance to blood flow. Normally, largeepicardial vessel resistance (R1) is trivial until atheroscleroticobstructions compromise the lumen. Precapillary arterioles (R2), 100 to500 μM in size) are resistive vessels connecting epicardial tomyocardial capillaries and are the principal controllers of coronaryblood flow. They contribute approximately 25% to 35% of total coronaryresistance. Distal precapillary arteriolar vessels (<100 μm indiameter), the main site of metabolic regulation of coronary blood flow,are responsible for 40-50% of coronary flow resistance. The densenetwork of about 4000 capillaries per square millimeter ensures thateach myocyte is adjacent to a capillary. Capillaries are not uniformlypatent (meaning open; affording free passage), because precapillarysphincters regulate flow according to the needs of the myocardium.

Several conditions, such as left ventricular hypertrophy, myocardialischemia, or diabetes, can impair the microcirculatory resistance (R3),blunting the maximal absolute increase in coronary flow in times ofincreased oxygen demand.

Ischemia

The myocardium depends almost entirely on aerobic metabolism, sinceoxygen stores in the heart are meager. Myocardial oxygen supply risesand falls in response to the oxygen (energy) demands of the myocardium.The term “autoregulation” refers to the ability to maintain myocardialperfusion at constant levels in the face of changing driving forces.Autoregulation maintains coronary perfusion at relatively constantlevels over a wide range of mean aortic pressure. When aortic pressureexceeds its upper or lower limits, coronary blood flow precipitouslydeclines or increases proportionately.

The heart needs to be supplied with a sufficient quantity of oxygen toprevent underperfusion. When reduced perfusion pressure distal tostenoses is not compensated by autoregulatory dilation of the resistancevessels, ischemia, meaning a lack of blood supply and oxygen, occurs.Because the zone least supplied generally is the farthest out, ischemiagenerally appears in areas farthest away from the blood supply.

After total or near-total occlusion of a coronary artery, myocardialperfusion occurs by way of collaterals, meaning vascular channels thatinterconnect epicardial arteries. Collateral channels may form acutelyor may preexist in an under-developed state before the appearance ofcoronary artery disease. Preexisting collaterals are thin-walledstructures ranging in diameter from 20 μm to 200 μm, with a variabledensity among different species. Preexisting collaterals normally areclosed and nonfunctional, because no pressure gradient exists to driveflow between the arteries they connect. After coronary occlusion, thedistal pressure drops precipitously and preexisting collaterals openvirtually instantly.

The term “myocardial ischemia” refers to a decrease in blood supply andoxygen to the cells of the myocardium. The development of myocardialischemia has been attributed to two mechanisms: (1) increased myocardialoxygen demand, and (2) decreased myocardial perfusion and oxygendelivery. (Willerson, J. T. et al., J. Am. Coll. Cardiol. 8(1): 245-50(1986)). Myocardial ischemia generally appears first and is moreextensive in the subendocardial region, since these deeper myocardiallayers are farthest from the blood supply, with greater need for oxygen.

Transient ischemia, hibernating myocardium, and myocardial infarctionare clinically different conditions.

Transient Ischemia. The term “transient ischemia” as used herein refersto a reversible (meaning that the myocytes survive the insult) narrowingof a coronary artery at rest or with exercise where there is no thrombusor plaque rupture but where blood supply cannot be met. Every time theheart's oxygen demand increases, an imbalance between oxygen demand andsupply is created. Transient ischemia produces a cascade of eventsbeginning with metabolic and biochemical alterations leading to impairedventricular relaxation and diastolic dysfunction, impaired systolicfunction, and electrocardiographic abnormalities with ST segmentalterations, followed by increased end-diastolic pressure with leftventricular dyssynchrony, hypokineses, akinesis, and dyskinesis, andlastly painful symptoms of angina. Even though ischemic myocytesexperience physiological and metabolic changes within seconds of thecessation of coronary flow, resulting in T wave and sometimes ST segmentabnormalities (but without serum enzyme elevation), no cell deathresults from the ischemia. Kloner, R. A. and Jennings, R B, Circulation104: 2981-89 (2001). Once blood flow is re-established, a completerecovery of myocyte contractile function takes place.

Although angina pectoris (chest pain) may be a symptom of transientischemia, by and large transient ischemia is silent (meaning ST-segmentdepression of at least 1 mm is present without associated symptoms,e.g., chest pain) in 79% of subjects. In most patients with stableangina, for example, physical effort or emotion, with a resultantincrease in heart rate, blood pressure, or contractile state, or anycombination thereof, increases myocardial oxygen demand without anadequate delivery in oxygen delivery through tightly narrowed (stenosed)coronary arteries. More than 40% of patients with stable angina treatedwith one or more antianginal drugs have frequent episodes of silentischemia, which has been shown to predict a higher risk of coronaryevents and cardiac death. Deedwania, P C, Carbajal, E V, Arch. Intern.Med. 150: 2373-2382 (1991).

Chronic Myocardial Ischemia. The term “chronic myocardial ischemia(CMI)” as used herein refers to a prolonged subacute or chronic state ofmyocardial ischemia due to narrowing of a coronary blood vessel in whichthe myocardium “hibernates”, meaning that the myocardium downregulatesor reduces its contractility, and hence its myocardial oxygen demand, tomatch reduced perfusion, thereby preserving cellular viability andpreventing myocardial necrosis. This hibernating myocardium is capableof returning to normal or near-normal function on restoration of anadequate blood supply. Once coronary blood flow has been restored tonormal or near normal and ischemia is resolved, however, the hibernatingmyocardium still does not contract. This flow-function mismatchresulting in a slow return of cardiac function after resolution ofischemia has been called stunning. The length of time for function toreturn is quite variable, ranging from days to months, and is dependenton a number of parameters, including the duration of the originalischemic insult, the severity of ischemia during the original insult,and the adequacy of the return of the arterial flow. A number of studieshave provided evidence for inflammation in hibernating myocardium.Heusch, G. et al., Am. J. Physiol. Heart Circ. Physiol. 288: 984-99(2005). A study conducted in a porcine model of myocardial hibernationin which the mean rest (left anterior descending coronary artery (LAD)coronary blood flow was reduced to about 60% of baseline for a period of24 hours to four weeks, detected apoptotic myocytes in all experimentalpigs in the hibernating regions supplied by the stenotic LAD, suggestingthat functional downregulation may not be adequate to prevent gradual,ongoing myocyte death through apoptosis in hibernating myocardium. Chen,C, et al., J. Am. Coll. Cardiol. 30: 1407-12 (1997).

Acute Myocardial Infarction (AMI). Another type of insult occurs duringAML AMI is an abrupt change in the lumen of a coronary blood vesselwhich results in ischemic infarction, meaning that it continues untilheart muscle dies. On gross inspection, myocardial infarction can bedivided into two major types: transmural infarcts, in which themyocardial necrosis involves the full or nearly full thickness of theventricular wall, and subendocardial (nontransmural) infarcts, in whichthe myocardial necrosis involves the subendocardium, the intramuralmyocardium, or both, without extending all the way through theventricular wall to the epicardium. There often is total occlusion ofthe vessel with ST segment elevation because of thrombus formationwithin the lumen as a result of plaque rupture. The prolonged ischemicinsult results in apoptotic and necrotic cardiomyocyte cell death. SeeKajstura, J., et al., Lab Invest. 74: 86-107 (1996). Necrosiscompromises the integrity of the sarcolemmal membrane and intracellularmacromolecules such that serum cardiac markers, such as cardiac-specifictroponins and enzymes, such as serum creatine kinase (CK), are released.In addition, the patient may have electrocardiogram (ECG) changesbecause of full thickness damage to the muscle. An ST-ElevationMyocardial Infarction (STEMI) is a larger injury than a non-ST-elevationmyocardial infarction. ST-segment elevation and Q waves on the ECG, twofeatures highly indicative of myocardial infarction, are seen in onlyabout half of myocardial infarction cases on presentation.

AMI remains common with a reported annual incidence of 1.1 million casesin the United States alone (Antman, E. M., Braunwald, E., AcuteMyocardial Infarction, in Principles of Internal Medicine, 15th Ed.,Braunwald, E. et al., Eds., New York: McGraw-Hill (2001)). Preclinicaland clinical data demonstrate that following a myocardial infarction,the acute loss of myocardial muscle cells and the accompanyingperi-infarct border zone hypo-perfusion result in a cascade of eventscausing an immediate diminution of cardiac function, with the potentialfor long term persistence. The extent of myocardial cell loss isdependent on the duration of coronary artery occlusion, existingcollateral coronary circulation and the condition of the cardiacmicrovasculature. Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer,M. A., Braunwald, E., Circulation 81: 1161-72 (1990); Sheilban, I. e.al., J. Am. Coll. Cardiol. 38: 464-71 (2001); Braunwald E., Bristow, M.R., Circulation 102: IV-14-23 (2000); Rich et al., Am. J. Med. 92:7-13(1992); Ren et al., J. Histochem. Cytochem. 49: 71-79 (2002); Hirai, T.et al., Circulation 79: 791-96 (1989); Ejiri, M. et al., J. Cardiology20: 31-37 (1990). Because myocardial cells have virtually no ability toregenerate, myocardial infarction leads to permanent cardiac dysfunctiondue to contractile-muscle cell loss and replacement with nonfunctioningfibrotic scarring. Frangogiannis, N. G., et al., Cardiovascular Res.53(1): 31-47 (2002). Moreover, compensatory hypertrophy of viablecardiac muscle leads to microvascular insufficiency that results infurther demise in cardiac function by causing myocardial musclehibernation and apoptosis of hypertrophied myocytes in the peri-infarctborder zone.

Among survivors of myocardial infarction, residual cardiac function isinfluenced by the extent of ventricular remodeling (meaning changes insize, shape, and function, typically a progressive decline in function,of the heart after injury). Alterations in ventricular topography(meaning the shape, configuration, or morphology of a ventricle) occurin both infarcted and healthy cardiac tissue after myocardialinfarction. Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72(1990). Ventricular dilatation (meaning a stretching, enlarging orspreading out of the ventricle) causes a decrease in global cardiacfunction and is affected by the infarct size, infarct healing andventricular wall stresses. Recent efforts to minimize remodeling havebeen successful by limiting infarct size through rapid reperfusion(meaning restoration of blood flow) using thromobolytic agents, andmechanical interventions, including, but not limited to, placement of astent, along with reducing ventricular wall stresses by judicious use ofpre-load therapies and proper after-load management. Id. Regardless ofthese interventions, a substantial percentage of patients experienceclinically relevant and long-term cardiac dysfunction after myocardialinfarction. Sheiban, I. et al., J. Am. Coll. Cardiol. 38: 464-71 (2001).Despite revascularization of the infarct related artery circulation andappropriate medical management to minimize ventricular wall stresses, asignificant percentage of these patients experience ventricularremodeling, permanent cardiac dysfunction, and consequently remain at anincreased lifetime risk of experiencing adverse cardiac events,including death. Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer,M. A., Braunwald, E., Circulation 81: 1161-72 (1990).

At the cellular level, immediately following a myocardial infarction,transient generalized cardiac dysfunction uniformly occurs. In thesetting of a brief (i.e., lasting three minutes to five minutes)coronary artery occlusion, energy metabolism is impaired, leading todemonstrable cardiac muscle dysfunction that can persist for up to 48hours despite immediate reperfusion. This so-called “stunned myocardiumphenomenon” occurs subsequent to or after reperfusion and is thought tobe a result of reactive oxygen species. The process is transient and isnot associated with an inflammatory response. Frangogiannis, N. G., etal., Cardiovascular Res. 53(1): 31-47 (2002). After successfulrevascularization, significant recovery from stunning occurs withinthree to four days, although complete recovery may take much longer.Soli, R., Prog. Cardiovascular Disease 40(6): 477-515 (1998); Sakata, K.et al., Ann. Nucleic Med. 8: 153-57 (1994); Wollert, K. C. et al.,Lancet 364: 141-48 (2004).

Coronary artery occlusion of more significant duration, i.e., lastingmore than five minutes, leads to myocardial ischemia (i.e. aninsufficient blood flow to the heart's muscle mass) and is associatedwith a significant inflammatory response that begins immediately afterreperfusion and can last for up to several weeks. Frangogiannis, N. G.,et al., Cardiovascular Res. 53(1): 31-47 (2002); Frangogiannis, N. G. etal., Circulation 98: 687-798 (1998).

The inflammatory process following reperfusion is complex. Initially itcontributes to myocardial damage but later leads to healing and scarformation. This complex process appears to occur in two phases. In thefirst so-called “hot” phase (within the first five days), reactiveoxygen species (in the ischemic myocardial tissue) and complementactivation generate a signal chemotactic for leukocytes (chemotaxis isthe directed motion of a motile cell, organism or part towardsenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent) and initiate a cytokine cascade. Lefer,D. J., Granger, D. N., Am. J. Med. 4:315-23 (2000); Frangogiannis, N.G., et al., Circulation 7:699-710 (1998). Mast cell degranulation, tumornecrosis factor alpha (TNFα) release, and increased interleukin-6(IL-6), intercellular adhesion molecule 1 (“ICAM-1” or CD-54, a receptortypically expressed on endothelial cells and cells of the immunesystem), selectin (L, E and P) and integrin (CD11a, CD11b and CD18)expression all appear to contribute to neutrophil accumulation anddegranulation in ischemic myocardium. Frangogiannis, N. G. et al.,Circulation 7: 699-710 (1998), Kurrelmeyer, K. M, et al., Proc. Natl.Acad. Sci USA. 10: 5456-61 (2000); Lasky, L. A., Science 258: 964-69(1992); Ma, X. L., et al., Circulation 88(2): 649-58 (1993); Simpson, P.J. et al., J. Clin. Invest. 2: 624-29 (1998). Neutrophils contributesignificantly to myocardial cell damage and death through microvascularobstruction and activation of neutrophil respiratory burst pathwaysafter ligand-specific adhesion to cardiac myocytes. Entman, M. L., etal., J. Clin. Invest. 4: 1335-45 (1992). During the “hot” phase,angiogenesis is inhibited due to the release of angiostatic substances,including interferon gamma-inducible protein (IP 10). Frangogiannis, N.G., et al., FASEB J. 15: 1428-30 (2001).

In the second phase, the cardiac repair process begins (about day 6 toabout day 14), which eventually leads to scar formation (about day 14 toabout day 21) and subsequent ventricular remodeling (about day 21 toabout day 90). Soon after reperfusion, monocytes infiltrate theinfarcted myocardium. Attracted by complement (C5a), transforming growthfactor B1 (“TGF-B1”) and monocyte chemotactic protein 1 (“MCP-1”),monocytes differentiate into macrophages that initiate the healingprocess by scavenging dead tissue, regulating extracellular matrixmetabolism, and inducing fibroblast proliferation. Birdshall, H. H., etal., Circulation 3: 684-92 (1997). Secretion of interleukin 10 (IL-10)by infiltrating lymphocytes also promotes healing by down-regulatinginflammatory cytokines and influencing tissue remodeling. Frangogiannis,N. G. et al., J. Immunol. 5:2798-2808 (2000). Mast cells also appear tobe involved in the later stages of myocardial repair by participating inthe formation of fibrotic scar tissue. Stem Cell Factor (SCF) is apotent attractor of mast cells. SCF mRNA has been shown to beup-regulated in ischemic myocardial segments in a canine model ofmyocardial infarction and thus may contribute to mast cell accumulationat ischemic myocardial sites. Franigogiannis, N. G. et al., Circulation98: 687-798 (1998). Mast cell products (including TGF-B, basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF) and gelatinases A and B) induce fibroblast proliferation,influence extracellular matrix metabolism, and induce angiogenesis.Fang, K. C., et al., J. Immunol. 162: 5528-35 (1999); Takeshi, S., etal., Cardiology 93: 168-74 (2000).

Following a myocardial infarction, neoangiogenesis occurs after the“hot” phase of the inflammatory process subsides (about day 5)coincident with rising levels of VEGF (VEGF peaks at about day 7 andgradually subsides to baseline at about day 14 to about day 21). Duringthis phase of the healing process, endothelial precursor cells (EPCs)are mobilized and recruited to the infarct site. Shinitani, S., et al.,Circulation 103: 2776-79 (2001). Without being limited by theory, it hasbeen suggested that the chemokine stromal cell derived factor-1 (SDF-1),which is the ligand for the CXCR-4 chemokine receptor expressed by CD34+cells, also plays a role in homing of cells to areas of ischemic damage.Ceredini, D. J., et al., Nature Medicine 10: 858-63 (2004); Askari, A.,et al., Lancet 362: 697-703 (2003); Yamaguchi, J. et al., Circulation107: 1322-34 (2003). While it is known that SDF-1 plays a role inhematopoiesis and is involved in migration, homing and survival ofhematopoietic progenitors, and while SDF-1 has been implicated inischemic neovascularization in vivo by augmenting EPC recruitment toischemic sites (Yamaguchi et al. Circulation 107:1322-1328 (2003),SDF-1's role in neoangiogenesis is not certain. There is suggestiveevidence implicating SDF-1. For example, SDF-1 gene expression isupregulated during hypoxia, a deficiency of oxygen in the tissues, byhypoxia inducible factor-1. Furthermore, CD34+ cells are capable ofhoming to areas of ischemia, rich in SDF-1, including infarctedmyocardium. Askari et al., Lancet 362: 697-703 (2003). Moreover,virtually all CD34+ CXCR-4+ cells co-express VEGF-2 and thereforemigrate in response to VEGF as well as SDF-1. Peichev M., et al., Blood95: 952-58 (2000). CD34+CXCR-4+VEGF-1 cells, once recruited, are capableof contributing to neoangiogenesis. Yamaguchi, J. et al., Circulation107: 1322-34 (2003).

The Peri-Infarct Border Zone

The zone of dysfunctional myocardium produced by coronary arteryocclusion extends beyond the infarct region to include a variableboundary of adjacent normal appearing tissue. (Hu, Q., et al., Am. J.Physiol. Heart Circ. Physiol. 291: H648-657 (2006)). This ischemic, butviable, perinfarct zone of tissue separates the central zone ofprogressive necrosis from surrounding normal myocardium. Theperi-infarct zone does not correlate with enzymatic parameters ofinfarct size and is substantially larger in small infarcts. Stork, A.,et al., European Radiol. 16(10): 2350-57 (2006).

Ischemia due to edema and compression of the blood vessels in the borderzone may be very important to outcome after an AMI. It is known, forexample, that after an AMI, transient ischemia occurs in the borderzones, and that percutaneous coronary interventions, which open up theinfarct-related artery, can adversely affect the health of theperi-infarct border zones. It has been suggested that intermediatelevels of mean blood flow can exist as the result of admixture ofpeninsulas of ischemic tissue intermingled with regions of normallyperfused myocardium at the border of an infarct. (Hu, Q., et al., Am. J.Physiol. Heart Circ Physiol. 291: H648-657 (2006)). However, theboundary of the intermingled coronary microvessels, which in dogs is nomore than 3 mm in width, cannot explain the relatively broad region ofdysfunctional myocardium surrounding an infarct. Murdock, R H, Jr., etal., Cir. Res. 52: 451-59 (1983); Buda, A J, et al., J. Am. Coll.Cardiol. 8: 150-58 (1986). Progressive dysfunction of this peri-infarctmyocardium over time may contribute to the transition from compensatedremodeling to progressive heart failure after an AMI.

Heart Failure

Heart failure is a complex clinical syndrome that arises secondary toabnormalities of cardiac structure and/or function that impair theability of the left ventricle to fill or eject blood. See Hunt, S. J.Am. Coll. Cardiol. 46: e1-e82 (2005). It is a progressive conditionwhere the heart muscle weakens and cannot pump blood efficiently.Patients may be categorized as having heart failure with depressedejection fraction (“EF”) (referred to as “systolic failure”), or havingheart failure with a normal EF or heart failure with a preserved EF(referred to as “diastolic failure”). Patients may have significantabnormalities of left ventricle (LV) contraction and relaxation and yethave no symptoms, in which case they are referred to as having“asymptomatic heart failure”. When a patient with chronic heart failuredeteriorates, the patient is referred to as having “decompensated heartfailure”, or, if the symptoms arise abruptly, as having “acutedecompensated heart failure”.

The various diagnostic criteria used to determine the presence of heartfailure are shown in the following Table (V. L. Roger, Intl. J. Environ.Res. Public Health 7(4): 1807-30 (2010)):

European Society Gothenburg Score⁴ Framingham¹ Boston² of Cardiology³ nCriteria/method of assessment MAJOR CATEGORY I: 1. Symptoms of CARDIACSCORE CRITERIA: History heart failure (at rest History of heart Self-Paroxysmal Rest dyspnea or during exercise) disease (1-2 pts) reportnocturnal dyspnea (4 pts) and Angina (1-2 pts) Self- or orthopneaOrthopnea (4 pts) 2. Objective report Neck vein Paroxysmal evidence ofcardiac Edema (1 pt) Self- distension nocturnal dysfunction (at rest)report Rales dyspnea (3 pts) and Nocturnal Dyspnea Self- CardiomegalyDyspnea on 3. Response to (1 pt) report Acute pulmonary walking on leveltreatment directed Rales (1 pt) Physical edema S3 gallop (2 pts) towardsheart failure exam Increased venous Dyspnea on (in cases where Atrialfibrillation ECG pressure ≧16 cm climbing (1 pt) diagnosis is in (1 pt)water CATEGORY doubt). PULMONARY SCORE Circ. time ≧25 sec II: PhysicalCriteria 1 and 2 History of Chronic Self- Hepatojugular examinationshould be fulfilled in bronchitis/asthma report reflux Heart rate allcases (1-2 pts) MINOR abnormality (1-2 Cough, phlegm, or Self- CRITERIA:pts) wheezing (1 pt) report Ankle edema Jugular venous Rhonchi (2 pts)Physical Night cough pressure exam Dyspnea on elevation (1-2 Cardiac andpulmonary score are exertion pts) calculated and used to HepatomegalyLung crackles differentiate Cardiac form Pleural effusion (1-2 pts)pulmonary dyspnea Vital capacity Wheezing (3 pts) decreased 1/3 Thirdheart from maximum sound (3 pts) Tachycardia rate CATEGORY of ≧120/min)III: Chest MAJOR OR radiography MINOR Alveolar CRITERION: pulmonaryWeight loss ≧4.5 kg edema (4 pts) in 5 days in Interstitial response topulmonary treatment edema (3 pts) HEART Bilateral pleural FAILURE:effusions (3 pts) present with 2 Cardiothoracic major or 1 major ratio≧0.50 (3 and 2 minor pts) criteria Upper-zone flow redistribution (2pts) HEART FAILURE: Definite 8-12 pts, possible 5-7 pts, unlikely 4 ptsor less ¹McKee PA, Castelli WP, McNamara PM, Kannel WB. The naturalhistory of congestive heart failure: the Framingham study. N. Engl. J.Med 285: 1441-1446 (1971) ²Carlson K J, Lee DC Goroll AH, Lehy M,Johnson RA, an analysis of physicians' reasons for prescribing long-termdigitalis therapy in outpatients. J. Chronic Dis. 38: 733-39 (1985)³Guidelines for the diagnosis of heart failure The Task Force on HeartFailure of the European Society of Cardiology. Eur. Heart J. 16: 741-751(1995) ⁴Eriksson H, Caidahl K, Larsson B, Ohlson LO, Welin L, WilhelmsenL, Svardsudd K. Cardiac and pulmonary causes of dyspnoea-validation of ascoring test for clinical-epidemiological use: the Study of Men Born in1913. Eur. Heart J. 8: 1007-1014 (1987)[

The prognosis of heart failure is poor with reported survival estimatesof 50% at 5 years and 10% at 10 years; left ventricular dysfunction isassociated with an increase in the risk of sudden death. Id.

To date, no ideal therapy exists for preventing the long term adverseconsequences of vascular insufficiency, particularly vascularinsufficiency after myocardial infarction. Large vesselrevascularization (meaning the successful placement of a stent) isinsufficient in addressing increased demands posed by compensatorymyocardial hypertrophy. As a result, infarct extension and fibrousreplacement commonly occur, regardless of large vesselrevascularization, appropriate medical management of ventricular wallstresses, and potential natural, albeit suboptimal, CD34+ cell-mediatedneoangiogenesis (one of theories relating to the underlying cause ofmyocardial infarction is that the ability to mobilize these cells may bebiologically limited).

Intense interest has developed in evaluating the ability of endothelialand myocardial precursor cells to limit damage to the myocardium afterinfarction and to limit or prevent ventricular remodeling. Significantpreclinical data and some clinical data demonstrate the safety andpotential of cell therapy using a variety of cell precursors(particularly hematopoietic cells) to contribute to neoangiogenesis,limited cardiac myogenesis (principally by fusion), and musclepreservation in the myocardial infarct zone. See, e.g., Jackson, et al.,J. Clin. Invest. 107: 1395-1402 (2001); Edelberg, J. M., et al., Cir.Res. 90: e89-e93 (2002); Schichinger, V. et al., New Engl. J. Med. 355(12): 1210-21 (2006) (using bone marrow-derived progenitor cells);Assmus, B. et al., New Engl. J. Med. 355 (12) 1222-32 (2006) (using bonemarrow-derived progenitor cells), but see Lunde, K. et al., New Eng. J.Med. 355 (12): 1199-209 (2006) (using hone marrow-derived progenitorcells).

Bone marrow consists of a variety of precursor and mature cell types,including hematopoietic cells (the precursors of mature blood cells) andstromal cells (the precursors of a broad spectrum of connective tissuecells), both of which appear to be capable of differentiating into othercell types. Wang, J. S. et al., J. Thorn. Cardiovasc. Surg. 122: 699-705(2001); Tomita, S. et al., Circulation 100 (Suppl. II): 247-256 (1999);Saito, T. et al., Tissue Eng. 1: 327-43 (1995). Unmodified (i.e., notfractionated) marrow or blood-derived cells have been used in severalclinical studies, for example, Hamann, K. et al., Japan Cir. J. 65:845-47 (2001); Strauer, B. E., et al., Circulation 106: 1913-18 (2002);Assmus, et al., Circulation 106: 3009-3017 (2002); Dobert, N. et al.,Eur. J. Nuel. Med. Mal. Imaging, 8: 1146-51 (2004); Wollert, K. C. etal., Lancet 364: 141-48 (2004). Since the mononuclear fraction of bonemarrow contains stromal cells, hematopoietic precursors, and endothelialprecursors, the relative contribution of each of these populations tothe observed effects, if any, remains unknown.

CD34 is a hematopoietic stem cell antigen selectively expressed onhematopoietic stem and progenitor cells derived from human bone marrow,blood and fetal liver. Yin et al., Blood 90: 5002-5012 (1997); Miaglia,S. et al., Blood 90: 5013-21 (1997). Cells that express CD34 are termedCD34+. Stromal cells do not express CD34 and are therefore termed CD34-.CD34+ cells isolated from human blood may be capable of differentiatinginto cardiomyocytes, endothelial cells, and smooth muscle cells in vivo.See Yeh, et al., Circulation 108: 2070-73 (2003). CD34+ cells representapproximately 1% of bone marrow derived nucleated cells; CD34 antigenalso is expressed by immature endothelial cell precursors (matureendothelial cells do not express CD34). Peichev, M. et al., Blood 95:952-58 (2000). In vitro, CD34+ cells derived from adult bone marrow giverise to a majority of the granulocyte/macrophage progenitor cells(CFU-GM), some colony-forming units-mixed (CFU-Mix) and a minorpopulation of primitive erythroid progenitor cells (burst forming units,erythrocytes or BFU-E). Yeh, et al., Circulation 108: 2070-73 (2003).CD34+ cells also may have the potential to differentiate into, or tocontribute to, the development of new myocardial muscle, albeit at lowfrequency.

Techniques have been developed using immunomagnetic bead separation toisolate a highly purified and viable population of CD34+ cells from bonenarrow mononuclear cells. See U.S. Pat. Nos. 5,536,475, 5,035,994,5,130,144, 4,965,205, the contents of each of which is incorporatedherein by reference. Two clinical studies support the clinicalapplication of bone marrow derived CD34+ cells after myocardialinfarction. See C. Stamm, et al., Lancet 361: 45-46 (2003); Herenstein,B. et al., Blood Supplement, Abs. 2696 (2004).

Animal Models

Peripheral artery disease (PAD), also called peripheral vascular disease(PVD), is modeled by the hind limb model of ischemia in which thefemoral artery of the mouse is tied off to simulate peripheral arterydisease. PAD, which commonly affects the arteries supplying the leg andincludes all diseases caused by the obstruction of large arteries in thearms and legs, can result from atherosclerosis, inflammatory processesleading to stenosis, an embolism or thrombus formation. Restriction ofblood flow due to arterial stenosis or occlusion often leads patients tocomplain of muscle pain on walking (intermittent claudication). Anyfurther reduction in blood flow causes ischemic pain at rest. Thiscondition is called chronic limb ischemia, meaning the demand for oxygencannot be sustained when resting. Ulceration and gangrene may thensupervene in the toes, which are the furthest away from the bloodsupply, and can result in loss of the involved limb if not treated.

Therapies for limb ischemia have the goals of collateral development andblood supply replenishment. Bone marrow derived CD34+mononuclear cellshave been tested in such hindlimb ischemia models, but the hindlimbischemia model does not model what takes place in the heart. A preferredtherapy after AMI would stop cells from dying during recovery that leadsto reverse remodeling and failure, or replace the dying cells withcardiomyocytes.

The closest animal model, the pig model, is not a good model of humandisease because (i) all experiments generally are done innonatherosclerotic animals, (ii) the animals are not treated withangioplasty, (iii) normal pigs do not embolize blood vessels; (iv)circulation of the pig is not exactly the same as human; and (iv) theperi-infarct border zone may not be the same.

A marginal improvement in angina symptoms recently was reported whenCD34+ cells were mobilized with G-CSF, apheresed after 5 days, and theninjected into an ischemic area of the heart based on Naga mapping.[Northwestern University (2009, April 1). Adult Stem Cell Injections MayReduce Pain And Improve Walking In Severe Angina Patients. ScienceDaily.Retrieved Oct. 21, 2010, fromhttp://www.sciencedaily.com-/releases/2009/03/090330091706.htm] Datafrom a phase I trial conducted by the present inventors has providedevidence that subjects treated with at least 10×10⁶ isolated autologousCD34+hematopoietic stem cells containing a subpopulation of at least0.5×10⁶ potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediatedchemotactic activity (n=9) experienced significant improvement inresting perfusion rates at 6 months compared to subjects receiving 5million cells (n=6) and control (n=15), as measured by the SPECT TotalSeverity Score (−256 versus +13, p=0.01). U.S. Patent Applications61/169,850 and 61/119,552, incorporated herein by reference.

The described invention is a therapy for preventing the long-termadverse consequences of vascular insufficiency, particularly vascularinsufficiency that produces expansion of the myocardial infarct areaafter an AMI progressing to heart failure. It is proposed thatadministration of a potent dose of a nonexpanded, isolated population ofautologous mononuclear cells enriched for CD34+ cells, which furthercontain a subpopulation of potent CD34+ cells expressing CXCR-4 andhaving CXCR-4-mediated chemotactic activity administered early or lateafter occurrence of an AMI can result in a reduction in major adversecardiac events, including, but not limited to, premature death,recurrent myocardial infarction, the development of congestive heartfailure, significant arrhythmias, and acute coronary syndrome, and theworsening of congestive heart failure, significant arrhythmias, andacute coronary syndrome.

SUMMARY OF THE INVENTION

The described invention provides progressive compositions and methods totreat adverse consequences of a progressive myocardial injury due to avascular insufficiency. According to some embodiments, the vascularinsufficiency occurs early after an acute myocardial infarctionresulting from underlying disease. According to some embodiments, thevascular insufficiency occurs late after an acute myocardial infarctionresulting from underlying disease.

According to one aspect, the described invention provides a method oftreating a progressive myocardial injury due to a vascularinsufficiency, the method comprising the steps: (a) acquiring a sterilenonexpanded, isolated population of autologous mononuclear cellscomprising CD34+ cells, which further contain a subpopulation of potentCD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity froma subject under sterile conditions; (b) sterilely enriching the CD34+cells which further contain a subpopulation of potent CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity from the sterilenonexpanded, isolated population of autologous mononuclear cellscomprising CD34+ cells, wherein the enriched CD34+ cells which furthercontain a subpopulation of potent CD34+/CXCR-4+ cells that haveCXCR-4-mediated chemotactic activity are a chemotactic hematopoieticstem cell product; (c) administering parenterally through a catheter ona plurality of infusion dates during lifetime of subject a sterilepharmaceutical composition, the sterile pharmaceutical compositioncomprising: (i) a therapeutically effective amount of the sterilechemotactic hematopoietic stem cell product, wherein the therapeuticallyeffective amount of the chemotactic hematopoietic stem cell productcomprises at least 10×10⁶ CD34+ cells which further contain asubpopulation of at least 0.5×10⁶ potent CD34+ cells expressing CXCR-4and having CXCR-4 mediated chemotactic activity; and (ii) a stabilizingamount of serum, wherein the stabilizing amount of serum is greater than20% (v/v), wherein the chemotactic hematopoietic stem cell product isfurther characterized as having the following properties for at least 24hours when tested in vitro after passage through a catheter: (1) retainsthe CXCR-4-mediated activity of the chemotactic hematopoietic stem cellproduct; (2) at least 70% of the cells are CD34+ cells; (3) is at least70% viable; and (4) is able to form hematopoietic colonies in vitro; (d)optionally administering the chemotactic hematopoietic stem cell productat a plurality of infusion dates during the subject's lifetime; and (e)treating at least one adverse consequence of the progressive vascularinsufficiency.

According to one embodiment, step (a) further comprises freezing atleast one aliquot of the nonexpanded, isolated population of autologousmononuclear cells containing CD34+ cells, which further contain asubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity at −86° C. and cryostoring the at least one aliquotin the vapor phase of a liquid nitrogen freezer.

According to another embodiment, step (a) further comprises (i) thawingthe at least one aliquot of the frozen sterile nonexpanded, isolatedpopulation of autologous mononuclear cells containing CD34+ cells whichfurther contain a subpopulation of potent CD34+/CXCR-4+ cells that haveCXCR-4-mediated chemotactic activity; (ii) enriching the sterilenonexpanded, isolated population of autologous mononuclear cells forCD34+ cells, which further contain a subpopulation of potentCD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity,wherein the sterile nonexpanded, isolated population of autologousmononuclear cells enriched for CD34+ cells, which further contain asubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is a thawed sterile ehemotactic hematopoietie stemcell product; and (iii) administering to the subject on a secondinfusion date a therapeutically effective amount of the sterile thawedsterile chemotactic hematopoietic stem cell product, comprising (a) atleast 10×10⁶ CD34+ cells, which further contain a subpopulation of atleast 0.5×10⁶ potent CD34+ cells expressing CXCR-4 and having CXCR-4mediated chemotactic activity; and (b) a stabilizing amount of serum,wherein the stabilizing amount of serum is greater than 20% (v/v),wherein the sterile thawed chemotactic hematopoietic stem cell productis further characterized as having the following properties for at least24 hours following thawing of the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of potent CD34+/CXCR-4-+ cells that haveCXCR-4-mediated chemotactic activity when tested in vitro after passagethrough a catheter: (1) retains the CXCR-4-mediated activity of thesubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity; (2) at least 70% of the cells are CD34+ cells; (3)is at least 70% viable; and (4) is able to form hematopoietic coloniesin vitro.

According to another embodiment, enriching step (ii) occurs from atleast 1 day to at least 40 years after acquisition of the sterilenonexpanded, isolated population of autologous mononuclear cellscomprising CD34+ cells from the subject.

According to another embodiment, the sterile chemotactic hematopoieticstem cell product is administered parenterally through a catheter to thesubject within about 48 hours to about 72 hours of thawing step (i).

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired early after an acute myocardialinfarction.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired after peak inflammatory cytokinecascade production in an infarcted area.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired late after an acute myocardialinfarction.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired from at least 15 days to at least 40years after occurrence of an acute myocardial infarction.

According to another embodiment, the sterile thawed chemotactichematopoietic stem cell product is further characterized as having thefollowing properties for at least 48 hours following thawing of thenonexpanded, isolated population of autologous mononuclear cells whentested in vitro after passage through a catheter: (i) is able to formhematopoietic colonies; and (ii) retains at least 2% of theCXCR-4-mediated activity of the subpopulation of potent CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity.

According to another embodiment, the sterile thawed chemotactichematopoietic stem cell product is further characterized as having thefollowing properties for at least 72 hours following thawing of thenonexpanded, isolated population of autologous mononuclear cells whentested in vitro after passage through a catheter: (i) is able to formhematopoietic colonies; and (ii) retains at least 2% of theCXCR-4-mediated activity of the subpopulation of potent CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity.

According to another embodiment, the vascular insufficiency is anischemia. According to another embodiment, the ischemia is a myocardialischemia. According to another embodiment, the ischemia is a transientischemia. According to another embodiment, the ischemia is a chronicmyocardial ischemia. According to another embodiment, the vascularinsufficiency is a vascular insufficiency after an acute myocardialinfarction resulting from underlying disease. According to anotherembodiment, the ischemia is a peri-infarct border zone ischemia.

According to another embodiment, a first infusion date comprises aspecific time interval defined by a first time and a second time,wherein the first time is after peak inflammatory cytokine cascadeproduction in an infarcted area and the second time is before myocardialscar formation in the infarcted area. According to another embodiment,the first time of the first infusion date is at least about 5 dayspost-infarction. According to another embodiment, the first time of thefirst infusion date is about 5 days post-infarction and the second timeis about 14 days post-infarction.

According to another embodiment, the method treats cardiomyocyte celldeath in the peri-infarct border zone, relative to controls. Accordingto another embodiment, the method treats hypoperfusion in theperi-infarct border zone, relative to controls. According to anotherembodiment, the method treats myocardial hibernation in the peri-infarctborder zone, relative to controls. According to another embodiment, themethod decreases infarct area, relative to controls. According toanother embodiment, wherein the method decreases infarct mass, relativeto controls.

According to another embodiment, the progressive myocardial injury is aprogressive decline in heart muscle function following the acutemyocardial infarction.

According to another embodiment, step (e) comprises treating at leastone adverse consequence of an acute myocardial infarction selected frompremature death, recurrent myocardial infarction, development ofcongestive heart failure, development of significant arrhythmias,development of acute coronary syndrome, worsening of congestive heartfailure, worsening of significant arrhythmias, and worsening of acutecoronary syndrome.

According to another embodiment, the progressive myocardial injury isheart failure.

According to another embodiment, the catheter is a flow controlcatheter.

According to another embodiment, the catheter is a balloon dilatationcatheter.

According to another embodiment, the catheter has an internal diameterof at least about 0.36 mm.

According to another embodiment, administering step (c) is through thecatheter into myocardium. According to another embodiment, administeringstep (c) is through the catheter intravascularly.

According to another embodiment, the pharmaceutical composition furtherincludes at least one compatible active agent. According to anotherembodiment, the active agent is selected from the group consisting of anangiotensin converting enzyme inhibitor, a beta-blacker, a diuretic, ananti-arrhythmic agent, a hematopoietic stem cell mobilizing agent, atyrosine kinase receptor agonist, an anti-anginal agent, a vasoactiveagent, an anticoagulant agent, a fibrinolytic agent, and ahypercholesterolemic agent.

According to another embodiment, the tyrosine kinase receptor agonist ishuman neuregulin 1.

According to another aspect, the described invention provides a regimenfor treating a progressive myocardial injury due to a vascularinsufficiency in a revascularized subject, which comprises (a)administering parenterally through a catheter on a plurality of infusiondates during lifetime of the subject a sterile pharmaceuticalcomposition comprising a sterile chemotactic hematopoietic stem cellproduct, wherein the sterile chemotactic hematopoietic stem cell productcomprises (i) a nonexpanded, isolated population of autologousmononuclear cells enriched for CD34+ cells, which further contain asubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, wherein the therapeutically effective amount ofthe chemotactic hematopoietic stem cell product comprises at least10×10⁶ CD34+ cells which further contain a subpopulation of at least0.5×10⁶ potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediatedchemotactic activity; and (ii) a stabilizing amount of serum, whereinthe stabilizing amount of serum is greater than 20% (v/v), wherein thechemotactic hematopoietic stem cell product is further characterized ashaving the following properties for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product whentested in vitro after passage through a catheter: (1) retains theCXCR-4-mediated activity of the subpopulation of potent CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity; (2) at least 70%of the cells are CD34+ cells; (3) is at least 70% viable; and (4) isable to form hematopoietic colonies in vitro; and (b) treating at leastone adverse consequence of the progressive vascular insufficiency.

According to one embodiment, the vascular insufficiency is an ischemia.According to another embodiment, the ischemia is a myocardial ischemia.According to another embodiment, the ischemia is a transient ischemia.According to another embodiment, the ischemia is a chronic myocardialischemia. According to another embodiment, the vascular insufficiency isa vascular insufficiency after an acute myocardial infarction resultingfrom underlying disease.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired early after occurrence of an acutemyocardial infarction.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired after peak inflammatory cytokinecascade production in an infarcted area.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired late after occurrence of an acutemyocardial infarction.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells, which furthercontain a subpopulation of CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity, is acquired from at least 15 days to at least 40years after occurrence of an acute myocardial infarction.

According to another embodiment, a first infusion date comprises aspecific time interval defined by a first time and a second time, andwherein the first time is after peak inflammatory cytokine cascadeproduction in an infarcted area and the second time is before myocardialscar formation in the infarcted area.

According to another embodiment, the first time of the first infusiondate is at least about 5 days post-infarction. According to anotherembodiment, the first time of the first infusion date is about 5 dayspost-infarction and the second time is about 14 days post-infarction.According to another embodiment, the first infusion date is at least 5days after occurrence of an acute myocardial infarction. According toanother embodiment, a second infusion date is at least 30 days afteroccurrence of an acute myocardial infarction.

According to another embodiment, the ischemia is a peri-infarct borderzone ischemia. According to another embodiment, step (b) comprisestreating cardiomyocyte cell death in the poi-infarct border zone,relative to controls. According to another embodiment, step (b)comprises treating hypoperfusion in the peri-infarct border zone,relative to controls.

According to another embodiment, step (b) comprises treating myocardialhibernation in the peri-infarct border zone, relative to controls.According to another embodiment, step (b) comprises decreasing infarctarea, relative to controls. According to another embodiment, step (b)comprises decreasing infarct mass, relative to controls.

According to another embodiment, step (b) comprises treating at leastone adverse consequence of the acute myocardial infarction selected frompremature death, recurrent myocardial infarction, development ofcongestive heart failure, development of significant arrhythmias,development of acute coronary syndrome, worsening of congestive heartfailure, worsening of significant arrhythmias, and worsening of acutecoronary syndrome.

According to another embodiment, the progressive myocardial injury is aprogressive decline in heart muscle function following the acutemyocardial infarction. According to another embodiment, the progressivemyocardial injury is heart failure.

According to another embodiment, the catheter is a flow controlcatheter. According to another embodiment, the catheter is a balloondilatation catheter. According to another embodiment, the catheter hasan internal diameter of at least about 0.36 mm.

According to another embodiment, the composition is administered throughthe catheter into myocardium. According to another embodiment, thecomposition is administered through the catheter intravascularly.

According to another embodiment, the pharmaceutical composition furtherincludes at least one compatible active agent. According to anotherembodiment, the active agent is selected from the group consisting of anangiotensin converting enzyme inhibitor, a beta-blocker, a diuretic, ananti-arrhythmic agent, a hematopoietic stem cell mobilizing agent, atyrosine kinase receptor agonist, an anti-anginal agent, a vasoactiveagent, an anticoagulant agent, a fibrinolytic agent, and ahypercholesterolemic agent.

According to another embodiment, the tyrosine kinase receptor agonist ishuman neuregulin 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that the functional viability of the chemotactichematopoietic cell product of the invention at 72 hours is equivalent tothat at 48 hours.

FIG. 2 shows the migratory efficiency of the formulated chemotactichematopoietic stem cell product comprising CD34+ cells of the invention.

FIG. 3 shows the improved stability of CD34+ cells formulated in humanserum.

DETAILED DESCRIPTION

The present invention describes compositions and methods for preventingearly or late adverse consequences of vascular insufficiency, including,but not limited to, vascular insufficiency that occurs early or lateafter an acute myocardial infarction resulting from underlying disease.

Glossary

The term “administer” and its various grammatical forms as used hereinmeans to give or to apply. The term “administering” as used hereinincludes in vivo administration, as well as administration directly totissue ex vivo. Generally, compositions may be administered systemicallyeither parenterally or topically in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired, or may be locally administered by means suchas, but not limited to, injection, implantation, grafting, topicalapplication, or parenterally. A means of administering cells mayinclude, but is not limited to, infusion.

As used herein, the term “aliquot” refers to a portion of a totalamount.

As used herein, the term “angiogenesis” refers to the process offormation and development of blood vessels.

The terms “apoptosis” or “programmed cell death” refer to a highlyregulated and active process that contributes to biologic homeostasiscomprised of a series of biochemical events that lead to a variety ofmorphological changes, including blebbing, changes to the cell membrane,such as loss of membrane asymmetry and attachment, cell shrinkage,nuclear fragmentation, chromatin condensation, and chromosomal DNAfragmentation, without damaging the organism.

The term “c-kit” refers to a protein on the surface of some cells thatbinds to stem cell factor (a substance that causes certain types ofcells to grow). Altered forms of this receptor may be associated withsome types of cancer.

The term “cardiac biomarkers” refers to enzymes, proteins and hormonesassociated with heart function, damage or failure that are used fordiagnostic and prognostic purposes. Different biomarkers have differenttimes that their levels rise, peak, and fall within the body, allowingthem to be used not only to track the progress of a heart attack but toestimate when it began and to monitor for recurrence. Some of the testsare specific for the heart while others also are elevated by skeletalmuscle damage. Current cardiac biomarkers include, but are not limitedto CK (creatine phosphokinase or creatine kinase) and CK-MB (creatinekinase-myoglobin levels (to help distinguish between skeletal and heartmuscle)), troponin (blood levels of troponin I or T will remain high for1-2 weeks after a heart attack; troponin generally is not affected bydamage to other muscles), myoglobin (to determine whether muscle,particularly heart muscle, has been injured), and BNP (brain natriureticpeptide) or NT-proBNP (N-terminal prohormone brain natriuretic peptide(to help diagnose heart failure and grade the severity of that heartfailure).

The term “cardiac catheterization” refers to a procedure in which acatheter is passed through an artery to the heart, and into a coronaryartery. This procedure produces angiograms (i.e., x-ray images) of thecoronary arteries and the left ventricle, the heart's main pumpingchamber, which can be used to measure pressures in the pulmonary artery,and to monitor heart function.

The term “CD34+ cells” as used herein refers to hematopoietic stem andprogenitor cells derived from human bone marrow that “are positive for”i.e., “express”, a hematopoietic stem cell antigen, at least asubpopulation of which express CXCR-4, and that can migrate to areas ofinjury. The chemotactic hematopoietic stem cell product of the describedinvention that is enriched for CD34+ cells does not co-express VEGF-2(<1%).

The term “CD38” refers to a protein marker present on macrophages,dendritic cells, and activated B and NK cells, which may mediate theadhesion between lymphocytes and endothelial cells.

The terms “CD45” and “common leukocyte antigen” refer to a proteintyrosine phosphatase (PTP) located in hematopoietic cells excepterythrocytes and platelets.

The term “CD59” refers to a glycosylphosphatidylinositol (GPI)-linkedmembrane glycoprotein which protects human cells fromcomplement-mediated lysis.

The term “CXCR-4” as used herein refers to a G-protein-linked chemokinereceptor.

The term “cytokine” as used herein refers to small soluble proteinsubstances secreted by cells which have a variety of effects on othercells. Cytokines mediate many important physiological functionsincluding growth, development, wound healing, and the immune response.They act by binding to their cell-specific receptors located in the cellmembrane, which allows a distinct signal transduction cascade to startin the cell, which eventually will lead to biochemical and phenotypicchanges in target cells. Generally, cytokines act locally. They includetype I cytokines, which encompass many of the interleukins, as well asseveral hematopoietic growth factors; type II cytokines, including theinterferons and interleukin-10; tumor necrosis factor (“TNF”)-relatedmolecules, including TNFα and lymphotoxin; immunoglobulin super-familymembers, including interleukin 1 (“IL-1”); and the chemokines, a familyof molecules that play a critical role in a wide variety of immune andinflammatory functions. The same cytokine can have different effects ona cell depending on the state of the cell. Cytokines often regulate theexpression of and trigger cascades of, other cytokines.

The term “colony stimulating factor” refers to a cytokine responsiblefor controlling the production of white blood cells. Types of colonystimulating factors include granulocyte colony stimulating factor(G-CSF), macrophage colony stimulating factor (M-CSF), and granulocytemacrophage colony stimulating factor (GM-CSF).

The term “hematopoietic stem cell” refers to a cell isolated from theblood or from the bone marrow that can renew itself, differentiate to avariety of specialized cells, mobilize out of the bone marrow into thecirculating blood, and can undergo programmed cell death (apoptosis). Insome embodiments of the described invention, hematopoietic stem cellsderived from human subjects express at least one type of cell surfacemarker, including, but not limited to, CD34, CD38, HLA-DR, c-kit, CD59,Sca-1, Thy-1, and/or CXCR-4, or a combination thereof.

“HLA-DR” refers to a human class II histocompatibility antigen presenton several cell types, including antigen-presenting cells, B cells,monocytes, macrophages, and activated T cells.

The term “interleukin” as used herein refers to a cytokine secreted bywhite blood cells as a means of communication with other white bloodcells.

The terms “VEGF-1” or “vascular endothelial growth factor-1” are usedinterchangeably herein to refer to a cytokine that mediates numerousfunctions of endothelial cells including proliferation, migration,invasion, survival, and permeability. VEGF is believed to be criticalfor angiogenesis.

The term “chemokine” as used herein refers to a class of chemotacticcytokines that signal leukocytes to move in a specific direction. Theterms “chemotaxis” or “chemotactic” refer to the directed motion of amotile cell or part along a chemical concentration gradient towardsenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent.

The term “complete blood count” (CBC) refers to a laboratory test thatprovides detailed information about the amount and the quality of eachof the blood cell types. It usually includes a measurement of each ofthe three major blood cells (red blood cells, white blood cells, andplatelets) and a measure of the hemoglobin and hematocrit. “Hemoglobin”(HGB) refers to the number of grams of hemoglobin in a deciliter ofblood (g/dL). Normal hemoglobin levels in healthy adult human subjectsare about 14 g/dL to about 18 g/dL for men and about 12 g/dL to about 16g/dL for women. As a rough guideline, hemoglobin generally should beabout one-third the hematocrit. “Red Blood Cell Count” (RBC) refers tothe total number of red blood cells in a quantity of blood. Normalranges in human subjects are about 4.5 million cells/mm³ to about 6.0million cells/mm³ for men and about 4.0 million cells/mm³ to about 5.5million cells/mm³ for women. “White Blood Cell Count” (WBC) refers tothe total number of while blood cells or leukocytes in a quantity ofblood. Normal ranges in human subjects are about 4.3×103 cells/mm³ toabout 10.8×103 cells/mm3. “Hematocrit” (HCT) refers to the proportion ofred blood cells as a percentage of total blood volume. A normalhematocrit for human subjects is about 40% to about 55% for men andabout 35% to about 45% for women.

The term “disease” or “disorder”, as used herein, refers to animpairment of health or a condition of abnormal functioning. The term“syndrome,” as used herein, refers to a pattern of symptoms indicativeof some disease or condition. The term “condition”, as used herein,refers to a variety of health states and is meant to include disordersor diseases caused by any underlying mechanism or disorder, injury, andthe promotion of healthy tissues and organs.

As used herein, the term “early” refers to being or occurring at or nearthe beginning of a period of time or series of events. As used herein,the term “late” refers to being or occurring at an advanced period oftime or stage of a series of events.

As used herein, the term “enriching” or “purifying” refers to increasingthe fraction of cells of one type over the fraction of that type in astarting preparation. Cells may be enriched using any of the variousmarkers expressed, or not expressed, on certain cells in combinationwith suitable separation techniques. Suitable separation techniquesinclude, but are not limited to, immunomagnetic bead separation,affinity chromatography, density gradient centrifugation, and flowcytometry.

As used herein, the term “nonexpanded” refers to not being increased oramplified in number of cells by in vitro culture.

As used herein, the term “inflammation” refers to a response toinfection and injury in which cells involved in detoxification andrepair are mobilized to the compromised site by inflammatory mediators.Inflammation often is characterized by a strong infiltration ofleukocytes at the site of inflammation, particularly neutrophils(polymorphonuclear cells). These cells promote tissue damage byreleasing toxic substances at the vascular wall or in uninjured tissue.

Regardless of the initiating agent, the physiologic changes accompanyingacute inflammation encompass four main features: (1) vasodilation, whichresults in a net increase in blood flow, is one of the earliest sphysical responses to acute tissue injury; (2) in response toinflammatory stimuli, endothelial cells lining the venules contract,widening the intracellular junctions to produce gaps, leading toincreased vascular permeability, which permits leakage of plasmaproteins and blood cells out of blood vessels; (3) inflammation often ischaracterized by a strong infiltration of leukocytes at the site ofinflammation, particularly neutrophils (polymorphonuclear cells). Thesecells promote tissue damage by releasing toxic substances at thevascular wall or in uninjured tissue; and (4) fever, produced bypyrogens released from leukocytes in response to specific stimuli.

During the inflammatory process, soluble inflammatory mediators of theinflammatory response work together with cellular components in asystemic fashion in the attempt to contain and eliminate the agentscausing physical distress. The terms “inflammatory” orimmuno-inflammatory” as used herein with respect to mediators refers tothe molecular mediators of the inflammatory process. These soluble,diffusible molecules act both locally at the site of tissue damage andinfection and at more distant sites. Some inflammatory mediators areactivated by the inflammatory process, while others are synthesizedand/or released from cellular sources in response to acute inflammationor by other soluble inflammatory mediators. Examples of inflammatorymediators of the inflammatory response include, but are not limited to,plasma proteases, complement, kinins, clotting and fibrinolyticproteins, lipid mediators, prostaglandins, leukotrienes,platelet-activating factor (PAF), peptides and amines, including, butnot limited to, histamine, serotonin, and neuropeptides, proinflammatorycytokines, including, but not limited to, interleukin-1, interleukin-4,interleukin-6, interleukin-S, tumor necrosis factor (TNF),interferon-gamma, and interleukin 12.

The term “in-date” refers to the time interval between completion ofacquiring a preparation comprising an enriched population of potentCD34+ cells from a subject under sterile conditions and initiatingsterilely purifying potent CD34+ cells from the preparation. The term“out-date” refers to the time interval between completion of acquiring apreparation comprising an enriched population of potent CD34+ cells froma subject under sterile conditions and infusing the formulatedpharmaceutical composition comprising a chemotactic hematopoietic cellproduct into the subject.

The terms “infuse” or “infusion” as used herein refer to theintroduction of a fluid other than blood into a blood vessel of asubject, including humans, for therapeutic purposes.

The “infusion solution” of the described invention without autologousserum contains phosphate buffered saline (PBS) supplemented with 25 USPunits/ml of heparin and 1% human serum albumin (HSA). In someembodiments, the infusion solution is supplemented with serum. In someembodiments, the serum is autologous.

The term “injury” refers to damage or harm caused to the structure orfunction of the body of a subject caused by an agent or force, which maybe physical or chemical. The term “vascular injury” refers to injury tothe vasculature (i.e., the vascular network, meaning the network ofblood vessels or ducts that convey fluids, such as, without limitation,blood or lymph). The term “myocardial injury” refers to injury to themyocardium of the heart.

The term “macrophage” as used herein refers to a mononuclear, activelyphagocytic cell arising from monocytic stem cells in the bone marrow.These cells are widely distributed in the body and vary in morphologyand motility. Phagocytic activity typically is mediated by serumrecognition factors, including certain immunoglobulins and components ofthe complement system, but also may be nonspecific. Macrophages also areinvolved in both the production of antibodies and in cell-mediatedimmune responses, particularly in presenting antigens to lymphocytes.They secrete a variety of immunoregulatory molecules.

The terms “microbe” or “microorganism” are used interchangeably hereinto refer to an organism too small to be seen clearly with the naked eye,including, but not limited to, microscopic bacteria, fungi (molds),algae, protozoa, and viruses.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “myocardial infarction” refers to death or permanent damage toheart muscle. Most heart attacks are caused by blockage of coronaryarteries that interrupts flow of blood and oxygen to the heart muscle,leading to death of heart cells in that area. The damaged heart muscleloses its ability to contract, leaving the remaining heart muscle tocompensate for the weakened area. The described invention includes stepsrelated to evaluating the suitability of subjects for treatmentaccording to the described invention by using tests to look at the size,shape, and function of the heart as it is beating, to detect changes tothe rhythm of the heart, and to detect and evaluate damaged tissues andblocked arteries. Examples of such tests include, but are not limitedto, electrocardiography, echocardiography, coronary angiography, andnuclear ventriculography. Cardiac biomarkers also are used to evaluatethe suitability of subjects for treatment according to the describedinvention.

The term “necrosis” refers to the premature death of cells and livingtissue induced by external factors, such as infection, toxins or trauma.Necrotic tissue undergoes chemical reactions different from those ofapoptotic tissue. Necrosis typically begins with cell swelling,chromatin digestion, disruption of the plasma membrane and of organellemembranes. Damage to the lysosome membrane can trigger release oflysosomal enzymes, destroying other parts of the cell. Late necrosis ischaracterized by extensive DNA hydrolysis, vacuolation of theendoplasmic reticulum, organelle breakdown and cell lysis. The releaseof intracellular content after plasma membrane rupture is the cause ofinflammation in necrosis. Released lysosomal enzymes can trigger a chainreaction of further cell death. Necrosis of a sufficient amount ofcontiguous tissue can result in tissue death or gangrene.

The term “perfusion” as used herein refers to the process of nutritivedelivery of arterial blood to a capillary bed in biological tissue.Perfusion (“F”) can be calculated with the formula F=((PA−Pv)/R) whereinPA is mean arterial pressure, Pv is mean venous pressure, and R isvascular resistance. Tissue perfusion can be measured in vivo, by, forexample, but not limited to, magnetic resonance imaging (MRI)techniques. Such techniques include using an injected contrast agent andarterial spin labeling (ASL) (wherein arterial blood is magneticallytagged before it enters into the tissue of interest and the amount oflabeling is measured and compared to a control recording).

The term “persisting” as used herein refers to that which isnever-ceasing or indefinitely continuous.

As used herein, the term “potent” or “potency” refers to the necessarybiological activity of the chemotactic hematopoietic stem cell productof the described invention, i.e., potent cells of the describedinvention remain viable, are capable of mediated mobility, and are ableto grow, i.e., to form hematopoietic colonies in an in vitro CFU assay.

The term “progenitor cell” as used herein refers to an immature cell inthe bone marrow that may be isolated by growing suspensions of marrowcells in culture dishes with added growth factors. Progenitor cellsmature into precursor cells that mature into blood cells. Progenitorcells are referred to as colony-forming units (CFU) or colony-formingcells (CFC). The specific lineage of a progenitor cell is indicated by asuffix, such as, but not limited to, CFU-E (erythrocytic), CFU-GM(granulocytic/macrophage), and CFU-GEMM (pluripotent hematopoieticprogenitor).

The term “progressive” as used herein refers to that which graduallyadvances in extent.

The term “repair” as used herein as a noun refers to any correction,reinforcement, reconditioning, remedy, making up for, making sound,renewal, mending, patching, or the like that restores function. Whenused as a verb, it means to correct, to reinforce, to recondition, toremedy, to make up for, to make sound, to renew, to mend, to patch or tootherwise restore function. In some embodiments “repair” includes fullrepair and partial repair.

The term “reverse” as used herein refers to a change to the contrary, orto a turning backward in nature or effect.

The term “Sea-1” or “stem cell antigen-1” refers to a surface proteincomponent in a signaling pathway that affects the self-renewal abilityof mesenchymal stem cells.

The term “stem cells” refers to undifferentiated cells having highproliferative potential with the ability to self-renew that can generatedaughter cells that can undergo terminal differentiation into more thanone distinct cell phenotype.

The term “stent” is used to refer to a small tube used to prop open anartery. The stent is collapsed to a small diameter, put over a ballooncatheter, inserted through a main artery in the groin (femoral artery)or arm (brachial artery) and threaded up to the narrowed/blocked sectionof the artery. When it reaches the right location, the balloon isinflated slightly to push any plaque out of the way and to expand theartery (balloon angioplasty). When the balloon is inflated, the stentexpands, locks in place and forms a scaffold to hold the artery open.The stent stays in the artery permanently. In certain subjects, a stentreduces the renarrowing that occurs after balloon angioplasty or otherprocedures that use catheters. A stent also may help restore normalblood flow and keep an artery open if it has been torn or injured by theballoon catheter. Reclosure (restenosis) may be a problem with the stentprocedure. Drug-eluting stents are stents coated with drugs that areslowly released. These drugs may help keep the blood vessel fromreclosing.

The terms “subject” and “patients” are used interchangeably herein andinclude animal species of mammalian origin, including humans.

The term “Thy-1” refers to the Ig superfamily cell surface glycoproteinThy-1 expressed on immune cells and neurons of rodents and humans, whichis hypothesized to function in cell adhesion and signal transduction inT cell differentiation, proliferation, and apoptosis.

As used herein the terms “treat” or “treating” are used interchangeablyto include abrogating, substantially inhibiting, slowing or reversingthe progression of a condition, substantially ameliorating clinical oraesthetical symptoms of a condition, substantially preventing theappearance of clinical or aesthetical symptoms of a condition, andprotecting from harmful or annoying stimuli. Treating further refers toaccomplishing one or more of the following: (a) reducing the severity ofthe disorder; (b) limiting development of symptoms characteristic of thedisorder(s) being treated; (c) limiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting recurrenceof the disorder(s) in patients that have previously had the disorder(s);and (e) limiting recurrence of symptoms in patients that were previouslyasymptomatic for the disorder(s).

The term “vascular insufficiency” refers to insufficient blood flow.

The described invention provides progressive myocardialinjury-preventing pharmaceutical compositions and methods to treat orprevent a progressive myocardial injury due to a vascular insufficiencythat occurs early or late. The terms “formulation” and “composition” areused interchangeably herein to refer to a product of the describedinvention that comprises all active and inert ingredients. The term“active” refers to the ingredient, component or constituent of thecompositions of the described invention responsible for the intendedtherapeutic effect. The terms “pharmaceutical formulation” or“pharmaceutical composition” as used herein refer to a formulation orcomposition that is employed to prevent, reduce in intensity, cure orotherwise treat a target condition or disease.

In one aspect of the described invention, the hematopoietic stem cellsof the described invention can migrate, meaning that they can move fromone place, location or area to another. In one embodiment, hematopoieticstem cell migration is driven by CXCR-4 chemotaxis.

Compositions

The progressive myocardial injury-preventing pharmaceutical compositionof the described invention comprises a chemotactic hematopoietic stemcell product, the chemotactic hematopoietic stem cell product comprisinga nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+CXCR-4+ cells that have chemotactic activity. In someembodiments, this chemotactic activity is mediated by SDF-1, and/orCXCR-4. According to some embodiments, the chemotactic hematopoieticstem cell product is prepared by isolating or purifying CD34+hematopoietic stem cells from bone marrow, umbilical cord blood,peripheral blood, mobilized peripheral blood, umbilical cord, or adiposetissue harvested from the subject. According to some embodiments, thechemotactic hematopoietic stem cell product is prepared by isolating orpurifying CD34+ hematopoietic stem cells from mobilized peripheralblood. Treatment with hematopoietic growth factors has been shown tocause a marked rise in the number of hematopoietic progenitor cells inthe peripheral blood as measured by the presence of CD34+ cells or asmeasured in a colony formation assay as CFUs. Such mobilized-peripheralblood hematopoietic stem cells (HSCs) have been used fortransplantation, immunotherapy, and cardiovascular regenerativemedicine. Colony stimulating factors, for example, are agents used forhematopoietic stem cell mobilization. Examples of colony stimulatingfactors include, without limitation, G-CSF, GM-CSF, and pharmaceuticallyacceptable analogs and derivatives thereof. For example, filgrastim, aG-CSF analog produced by recombinant technology, is marketed under thebrand names Neupogen® (Amgen); Religrast® (Reliance Life Sciences),Nugraf® (Zenotech Laboratories, Ltd., and Neukine® (IntasBiopharmaceuticals).

According to some embodiments, the nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells which furthercontain a subpopulation of potent CD34+/CXCR-4+ cells that haveCXCR-4-mediated chemotactic activity can be acquired from the subject atany time. According to some embodiments, the nonexpanded, isolatedpopulation of autologous mononuclear cells is acquired early after anAMI. According to some such embodiments, the nonexpanded, isolatedpopulation of autologous mononuclear cells is acquired 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days or more after the occurrence of an AMI. According to someembodiments, the nonexpanded, isolated population of autologousmononuclear cells comprising CD34+ cells which further contain asubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity is acquired late after the occurrence of an AMI.According to some such embodiments, the nonexpanded, isolated populationof autologous mononuclear cells is acquired at least 15 days, at least16 days, at least 17 days, at least 18 days, at least 19 days, at least20 days, at least 21 days, at least 22 days, at least 23 days, at least24 days, at least 25 days, at least 26 days, at least 27 days, at least28 days, at least 29 days, at least 30 days, at least 60 days, at least90 days, at least 120 days, at least 150 days, at least 180 days, ormore from the AMI. According to some embodiments, the nonexpanded,isolated population of autologous mononuclear cells comprising CD34+cells which further contain a subpopulation of potent CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity is acquired atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 7 months, at least 8months, at least 9 months, at least 10 months, at least 11 months, atleast 12 months, at least 16 months, at least 24 months, at least 30months, at least 36 months, at least 42 months, at least 48 months, atleast 54 months, at least 60 months, at least 66 months, at least 72months, at least 78 months, at least 84 months, at least 90 months, atleast 96 months, at least 102 months, at least 108 months, at least 114months, at least 120 months, at least 126 months, at least 132 months,at least 138 months, at least 144 months, at least 150 months, at least156 months, at least 162 months, at least 168 months, at least 174months, at least 180 months, at least 186 months, at least 192 months,at least 198 months, at least 204 months, at least 210 months, at least216 months, at least 222 months, at least 228 months, at least 234months, at least 240 months or more after occurrence of an AMI.According to some such embodiments, the nonexpanded, isolated populationof autologous mononuclear cells comprising CD34+ cells which furthercontain a subpopulation of potent CD34+/CXCR-4+ cells that haveCXCR-4-mediated chemotactic activity is acquired at least 3 years, 4years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years,12 years, 13 years, 14 years, 15 years, 16 years 17 years, 18 years, 19years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33years, 34 years, 35 years, 36 years 37 years, 38 years, 39 years, 40years or more after occurrence of an AMI. According to some embodiments,the nonexpanded, isolated population of autologous mononuclear cellscomprising CD34+ cells which further contain a subpopulation of potentCD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity, onceacquired, is frozen at −86° C. and cryostored in the vapor phase of aliquid nitrogen freezer as a plurality of aliquots for later usage.

According to the described invention, at least 70% of potent cells inthe nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areCD34+ cells. In some embodiments, at least 75% of cells in thenonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells containing a subpopulation of potent CD34+cells expressing CXCR-4 and having chemotactic activity are CD34+ cells.In some embodiments, at least 80% of potent cells in the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are CD34+ cells. Insome embodiments, at least 85% of potent cells in the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are CD34+ cells. Insome embodiments, at least 90% of potent cells in the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are CD34+ cells. Insome embodiments, at least 95% of potent cells in the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are CD34+ cells.

According to another embodiment, at least about 70% of the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are viable for atleast about 24 hours following acquisition of the chemotactichematopoietic stem cell product. According to another embodiment, atleast about 75% of the nonexpanded, isolated population of autologousmononuclear cells enriched for CD34+ cells, which further contain asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity are viable for at least about 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 80% of the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are viable for atleast 24 hours following acquisition of the chemotactic hematopoieticstem cell product. According to another embodiment, at least about 85%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least 24 hours following acquisition of the chemotactichematopoietic stem cell product. In some embodiments, at least about 90%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least 24 hours following acquisition of the chemotactichematopoietic stem cell product. In some embodiments, at least about 95%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least about 24 following acquisition of the chemotactichematopoietic stem cell product.

According to another embodiment, at least about 70% of the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are viable for atleast about 48 hours following acquisition of the chemotactichematopoietic stem cell product. According to another embodiment, atleast about 75% of the nonexpanded, isolated population of autologousmononuclear cells enriched for CD34+ cells, which further contain asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity are viable for at least about 48 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 80% of the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are viable for atleast 48 hours following acquisition of the chemotactic hematopoieticstem cell product. According to another embodiment, at least about 85%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least 48 hours following acquisition of the chemotactichematopoietic stem cell product. In some embodiments, at least about 90%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells containing a subpopulation of potent CD34+cells expressing CXCR-4 and having chemotactic activity are viable forat least 48 hours following acquisition of the chemotactic hematopoieticstem cell product. In some embodiments, at least about 95% of thenonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least about 48 following acquisition of the chemotactichematopoietic stem cell product.

According to another embodiment, at least about 70% of the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are viable for atleast about 72 hours following acquisition of the chemotactichematopoietic stem cell product. According to another embodiment, atleast about 75% of the nonexpanded, isolated population of autologousmononuclear cells enriched for CD34+ cells, which further contain asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity are viable for at least about 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 80% of the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity are viable for atleast 72 hours following acquisition of the chemotactic hematopoieticstem cell product. According to another embodiment, at least about 85%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least 72 hours following acquisition of the chemotactichematopoietic stem cell product. In some embodiments, at least about 90%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least 72 hours following acquisition of the chemotactichematopoietic stem cell product. In some embodiments, at least about 95%of the nonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity areviable for at least about 72 following acquisition of the chemotactichematopoietic stem cell product.

According to another embodiment, the nonexpanded, isolated population ofautologous mononuclear cells enriched for CD34+ cells, which furthercontain a subpopulation of potent CD34+ cells having chemotacticactivity can form hematopoietic colonies in vitro for at least about 24hours following acquisition from the subject of the chemotactichematopoietic stem cell product. According to another embodiment, thenonexpanded, isolated population of autologous mononuclear cellsenriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity canform hematopoietic colonies in vitro for at least about 48 hoursfollowing acquisition from the subject of the chemotactic hematopoieticstem cell product. According to another embodiment, the nonexpanded,isolated population of autologous mononuclear cells enriched for CD34+cells, which further contain a subpopulation of potent CD34+ cellsexpressing CXCR-4 and having chemotactic activity can form hematopoieticcolonies in vitro for at least about 72 hours following acquisition fromthe subject of the chemotactic hematopoietic stem cell product.

According to another embodiment, the progressive myocardialinjury-preventing composition further comprises at least about 10million isolated CD34+ cells acquired from the subject, which furthercontain a subpopulation of potent CD34+ cells expressing CXCR-4 andhaving CXCR-4-mediated chemotactic activity. According to anotherembodiment, the progressive myocardial injury-preventing compositionfurther comprises at least about 11 million isolated CD34+ cellsacquired from the subject, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity. According to another embodiment, the progressivemyocardial injury-preventing composition further comprises at leastabout 12 million isolated CD34+ cells acquired from the subject, whichfurther contain a subpopulation of potent CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity. According to anotherembodiment, the progressive myocardial injury-preventing compositionfurther comprises at least about 13 million isolated CD34+ cellsacquired from the subject, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity. According to another embodiment, the progressivemyocardial injury-preventing composition further comprises at leastabout 14 million isolated CD34+ cells acquired from the subject, whichfurther contain a subpopulation of potent CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity. According to anotherembodiment, the progressive myocardial injury-preventing compositionfurther comprises at least about 15 million isolated CD34+ cellsacquired from the subject, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity. According to another embodiment, the progressivemyocardial injury-preventing composition further comprises at leastabout 20 million isolated CD34+ cells acquired from the subject, whichfurther contain a subpopulation of potent CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity. According to anotherembodiment, the progressive myocardial injury-preventing compositionfurther comprises at least about 30 million isolated CD34+ cellsacquired from the subject, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity. According to another embodiment, the progressivemyocardial injury-preventing composition further comprises at leastabout 40 million isolated CD34+ cells acquired from the subject, whichfurther contain a subpopulation of potent CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity. According to anotherembodiment, the progressive myocardial injury-preventing compositionfurther comprises at least about 50 million isolated CD34+ cellsacquired from the subject, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity. According to another embodiment, the progressivemyocardial injury-preventing composition further comprises at leastabout 60 million isolated CD34+ cells acquired from the subject, whichfurther contain a subpopulation of potent CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity. According to anotherembodiment, the progressive myocardial injury-preventing compositionfurther comprises at least about 70 million isolated CD34+ cellsacquired from the subject, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity. According to another embodiment, the progressivemyocardial injury-preventing composition further comprises at leastabout 80 million isolated CD34+ cells acquired from the subject, whichfurther contain a subpopulation of potent CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity. According to anotherembodiment, the progressive myocardial injury-preventing compositionfurther comprises at least about 90 million isolated CD34+ cellsacquired from the subject, which further contain a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity. According to another embodiment, the progressivemyocardial injury-preventing composition further comprises at leastabout 100 million isolated CD34+ cells acquired from the subject, whichfurther contain a subpopulation of potent CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity.

For use in the present invention, CD34+ cells may be enriched/selectedby any techniques known to the skilled artisan. For example, in someembodiments, the isolated population of autologous mononuclear cellscomprising CD34+ cells is enriched for cells expressing CD34 cellantigen and CXCR-4 cell antigen by fluorescence activated cell sorting(FACS). In some embodiments, the isolated population of autologousmononuclear cells comprising CD34+ cells are enriched/selected bypositive or negative immunoseparation techniques. In some embodiments,isolation and/or purification of hematopoietic stem cells from theisolated population of autologous mononuclear cells comprising CD34+cells is based on cell fractionation methods based on size and celldensity, efflux of metabolic dyes, or resistance to cytotoxic agents. Inone embodiment, for example, the isolated population of autologousmononuclear cells comprising CD34+ cells in is enriched for CD34+cells/selected using a monoclonal anti-CD34 antibody and animmunomagnetic separation technique.

The isolated CD34+ cells may be identified, quantified and characterizedby techniques known in the art. For example, in some embodiments, thepercentage of CD34+ cells in the isolated population of autologousmononuclear cells comprising CD34+ cells and in the chemotactichematopoietic stem cell product can be determined by FACS analysis.According to another embodiment, CD34 protein expression is quantifiedby Western blot. The term “Western blot” refers to a method foridentifying proteins in a complex mixture; proteins are separatedelectrophoretically in a gel medium; transferred from the gel to aprotein binding sheet or membrane; and the sheet or membrane containingthe separated proteins exposed to specific antibodies which bind to,locate, and enable visualization of protein(s) of interest. For example,monoclonal anti-CD34 antibody can be used to detect CD34 protein adheredto a membrane in situ.

According to another embodiment, the expression of CD34 mRNA and DNA inthe isolated CD34+ cells may be quantified. The term “Northern blot” asused herein refers to a technique in which RNA from a specimen isseparated into its component parts on a gel by electrophoresis andtransferred to a specifically modified paper support so that the mRNA isfixed in its electrophoretic positions. CD34 related sequences areidentified using probes comprising a reporter molecule, such as, withoutlimitation, a radioactive label. According to another embodiment, thelevel of CD34 and/or CXCR-4 expression is/are determined by quantitativeor semi-quantitative PCR or real time PCR (“RT-PCR”) techniques. Theabbreviation “PCR” refers to polymerase chain reaction, which is atechnique for amplifying the quantity of DNA, thus making the DNA easierto isolate, clone and sequence. See, e.g., U.S. Pat. Nos. 5,656,493,5,333,675, 5,234,824, and 5,187,083, each of which is incorporatedherein by reference. Real-time PCR is a method of simultaneous DNAquantification and amplification,

whereby DNA is specifically amplified by polymerase chain reaction(PCR), and after each round of amplification, the DNA is quantified.

According to another embodiment, the isolated CD34+hematopoietic stemcells of the chemotactic hematopoietic stem cell product of thedescribed invention contain a subpopulation of CD34+ cells expressingCXCR-4 and having CXCR-4 mediated chemotactic activity. According toanother embodiment, the hematopoietic stem cell product of the describedinvention comprises a minimum number of isolated CD34+hematopoietic stemcells such that a subpopulation of at least 0.5×106 CD34+ cellsexpressing CXCR-4 and having CXCR-4 mediated chemotactic activity ispresent. According to another embodiment, at least about 2% of theCXCR-4 mediated chemotactic activity of the isolated CD34+ cellscontaining a subpopulation of potent CD34+ cells expressing CXCR-4 andhaving chemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 3% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 4% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 5% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 6% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 7% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 8% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 9% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 10% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 11% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 12% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 13% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 14% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 15% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 16% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 17% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 18% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 19% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 20% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 21% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 22% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 23% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 24% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 25% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 26% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 27% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 28% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 29% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 30% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 31% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 32% of the CXCR-4mediated chemotactic activity of the isolated. CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 33% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 34% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.

According to another embodiment, at least about 2% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 3% of the CXCR-4 mediated chemotacticactivity of the isolated CD34+ cells containing a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity isretained for at least 48 hours following acquisition of the enrichedpopulation of CD34+ cells. According to another embodiment, at leastabout 4% of the CXCR-4 mediated chemotactic activity of the isolatedCD34+ cells containing a subpopulation of potent CD34+ cells expressingCXCR-4 and having chemotactic activity is retained for at least 48 hoursfollowing acquisition of the enriched population of CD34+ cells.According to another embodiment, at least about 5% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 6% of the CXCR-4 mediated chemotacticactivity of the isolated CD34+ cells containing a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity isretained for at least 48 hours following acquisition of the enrichedpopulation of CD34+ cells. According to another embodiment, at leastabout 7% of the CXCR-4 mediated chemotactic activity of the isolatedCD34+ cells containing a subpopulation of potent CD34+ cells expressingCXCR-4 and having chemotactic activity is retained for at least 48 hoursfollowing acquisition of the enriched population of CD34+ cells.According to another embodiment, at least about 8% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 9% of the CXCR-4 mediated chemotacticactivity of the isolated CD34+ cells containing a subpopulation ofpotent CD34+ cells expressing CXCR-4 and having chemotactic activity isretained for at least 48 hours following acquisition of the enrichedpopulation of CD34+ cells. According to another embodiment, at leastabout 10% of the CXCR-4 mediated chemotactic activity of the isolatedCD34+ cells containing a subpopulation of potent CD34+ cells expressingCXCR-4 and having chemotactic activity is retained for at least 48 hoursfollowing acquisition of the enriched population of CD34+ cells.According to another embodiment, at least about 11% of the CXCR-4mediated chemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 12% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 13% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 14% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 15% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 16% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 17% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 18% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 19% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 20% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 21% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 22% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 23% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 24% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 25% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 26% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 27% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 28% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 29% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 30% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 31% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 32% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 33% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells. According toanother embodiment, at least about 34% of the CXCR-4 mediatedchemotactic activity of the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 48 hours followingacquisition of the enriched population of CD34+ cells.

According to another embodiment, at least about 2% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 3% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 4% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 5% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 6% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 7% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 8% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 9% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 10% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 11% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 12% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 13% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 14% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing, asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 15% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 16% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 17% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 18% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 19% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 20% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 21% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 22% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 23% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 24% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 25% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 26% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 27% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 28% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 29% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 30% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 31% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 32% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 33% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least about 34% of the CXCR-4mediated chemotactic activity of the isolated CD34⁺ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.

According to another embodiment, at least an average of about 17% of theCXCR-4 mediated chemotactic activity of the isolated CD34+ cellscontaining a subpopulation of potent CD34+ cells expressing CXCR-4 andhaving chemotactic activity is retained for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least an average of about 17% of theCXCR-4 mediated chemotactic activity of the isolated CD34+ cellscontaining a subpopulation of potent CD34+ cells expressing CXCR-4 andhaving chemotactic activity is retained for at least 48 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, at least an average of about 17% of theCXCR-4 mediated chemotactic activity of the isolated CD34+ cellscontaining a subpopulation of potent CD34+ cells expressing CXCR-4 andhaving chemotactic activity is retained for at least 72 hours followingacquisition of the chemotactic hematopoietic stem cell product.According to another embodiment, the isolated CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingchemotactic activity in the chemotactic hematopoietic cell productretain at least about 2% of the CXCR-4 mediated chemotactic activity forat least 72 hours following acquisition of the chemotactic hematopoieticstem cell product.

According to another embodiment, the pharmaceutical composition of theinvention further comprises serum at a concentration of at least 10%expressed as ml/100 cc final volume of the progressive myocardialinjury-preventing composition. According to another embodiment, thepharmaceutical composition of the invention further comprises serum at aconcentration of at least 11% expressed as ml/100 cc final volume of theprogressive myocardial injury-preventing composition. According toanother embodiment, the pharmaceutical composition of the inventionfurther comprises serum at a concentration of at least 12% expressed asml/100 cc final volume of the progressive myocardial injury-preventingcomposition. According to another embodiment, the pharmaceuticalcomposition of the invention further comprises serum at a concentrationof at least 13% expressed as ml/100 cc final volume of the progressivemyocardial injury-preventing composition. According to anotherembodiment, the pharmaceutical composition of the invention furthercomprises serum at a concentration of at least 14% expressed as ml/100cc final volume of the progressive myocardial injury-preventingcomposition. According to another embodiment, the pharmaceuticalcomposition of the invention further comprises serum at a concentrationof at least 15% expressed as ml/100 cc final volume of the progressivemyocardial injury-preventing composition. According to anotherembodiment, the pharmaceutical composition of the invention furthercomprises serum at a concentration of at least 16% expressed as ml/100cc final volume of the progressive myocardial injury-preventingcomposition. According to another embodiment, the pharmaceuticalcomposition of the invention further comprises serum at a concentrationof at least 17% expressed as ml/100 cc final volume of the progressivemyocardial injury-preventing composition. According to anotherembodiment, the pharmaceutical composition of the invention furthercomprises serum at a concentration of at least 18% expressed as ml/100cc final volume of the progressive myocardial injury-preventingcomposition. According to another embodiment, the pharmaceuticalcomposition of the invention further comprises serum at a concentrationof at least 19% expressed as ml/100 cc final volume of the progressivemyocardial injury-preventing composition. According to anotherembodiment, the pharmaceutical composition of the invention furthercomprises serum at a concentration of at least 20% expressed as ml/100cc final volume of the progressive myocardial injury-preventingcomposition. According to another embodiment, the minimum concentrationof serum present in the progressive myocardial injury-preventingcomposition is at least about 21% expressed as ml/100 cc final volume ofthe composition. According to another embodiment, the minimumconcentration of serum present in the progressive myocardialinjury-preventing composition is at least about 22% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 23% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 24% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 25% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 26% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 27% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 28% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 29% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 30% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 31% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 32% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 33% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 34% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 35% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 36% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 37% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 38% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 39% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 40% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 41% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 42% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 43% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 44% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 45% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 46% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 47% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 48% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 49% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 50% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 51% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 52% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 53% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 54% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 55% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 56% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 57% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 58% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 59% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 60% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 61% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 62% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 63% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 64% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 65% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 66% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 67% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 68% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the progressive myocardialinjury-preventing composition is at least about 69% expressed as ml/100cc final volume of the composition. According to another embodiment, theminimum concentration of serum present in the composition is at leastabout 70% expressed as ml/100 cc final volume of the composition.

According to another embodiment, the serum is autologous. According toanother embodiment, the serum is a synthetic or recombinant serum.

According to another embodiment, the maximum concentration of serumpresent in the progressive myocardial injury-preventing composition ofthe described invention is about 70% expressed as ml/100 cc final volumeof the composition. According to another embodiment, the maximumconcentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 69%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 68% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 67% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 66%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 65% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 64% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 63%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 62% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 61% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 60%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 59% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 58% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 57%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 56% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 55% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 54%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 53% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 52% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 51%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 50% expressed as ml/100 cc final volume of thecomposition.

According to another embodiment, the maximum concentration of serumpresent in the progressive myocardial injury-preventing composition ofthe described invention is about 49% expressed as ml/100 cc final volumeof the composition. According to another embodiment, the maximumconcentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 48%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 47% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 46% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 45%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 44% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 43% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 42%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 41% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 40% expressed as ml/100cc final volume of the composition.

According to another embodiment, the maximum concentration of serumpresent in the progressive myocardial injury-preventing composition ofthe described invention is about 39% expressed as ml/100 cc final volumeof the composition. According to another embodiment, the maximumconcentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 38%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 37% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 36% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 35%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 34% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 33% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 32%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 31% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 30% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 29%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 28% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 27% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 26%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 25% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 24% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 23%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 22% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 21% expressed as ml/100cc final volume of the composition. According to another embodiment, themaximum concentration of serum present in the progressive myocardialinjury-preventing composition of the described invention is about 20%expressed as ml/100 cc final volume of the composition. According toanother embodiment, the maximum concentration of serum present in theprogressive myocardial injury-preventing composition of the describedinvention is about 15% expressed as ml/100 cc final volume of thecomposition. According to another embodiment, the maximum concentrationof serum present in the progressive myocardial injury-preventingcomposition of the described invention is about 10% expressed as ml/100cc final volume of the composition.

In some embodiments, the progressive myocardial injury-preventingcomposition may be formulated with an excipient, carrier or vehicleincluding, but not limited to, a solvent. The terms “excipient”,“carrier”, or “vehicle” as used herein refers to carrier materialssuitable for formulation and administration of the chemotactichematopoietic stem cell product described herein. Carriers and vehiclesuseful herein include any such materials know in the art which arenontoxic and do not interact with other components. As used herein thephrase “pharmaceutically acceptable carrier” refers to any substantiallynon-toxic carrier useable for formulation and administration of thecomposition of the described invention in which the chemotactichematopoietic stem cell product of the described invention will remainstable and bioavailable.

The pharmaceutically acceptable carrier must be of sufficiently highpurity and of sufficiently low toxicity to render it suitable foradministration to the mammal being treated. It further should maintainthe stability and bioavailability of an active agent. Thepharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with an activeagent and other components of a given composition. For example, thepharmaceutically acceptable carrier may be, without limitation, abinding agent (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate,etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.), a disintegrant (e.g., starch, sodium starchglycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate,etc.). Other suitable pharmaceutically acceptable carriers for thecompositions of the described invention include, but are not limited to,water, salt solutions, alcohols, polyethylene glycols, gelatins,amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins,hydroxymethyleelluloses, polyvinylpyrrolidones and the like. Suchcarrier solutions also can contain buffers, diluents and other suitableadditives. The term “buffer” as used herein refers to a solution orliquid whose chemical makeup neutralizes acids or bases without asignificant change in pH. Examples of buffers envisioned by thedescribed invention include, but are not limited to, Dulbecco'sphosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water(D5W), and normal/physiologic saline (0.9% NaCl). In some embodiments,the infusion solution is isotonic to subject tissues. In someembodiments, the infusion solution is hypertonic to subject tissues.Compositions of the described invention that are for parenteraladministration may include pharmaceutically acceptable carriers such assterile aqueous solutions, non-aqueous solutions in common solvents suchas alcohols, or solutions in a liquid oil base.

In some embodiments, the carrier of the progressive myocardialinjury-preventing composition of the described invention may include arelease agent such as a sustained release or delayed release carrier. Insuch embodiments, the carrier may be any material capable of sustainedor delayed release of the active to provide a more efficientadministration, e.g., resulting in less frequent and/or decreased dosageof the composition, improve ease of handling, and extend or delayeffects on diseases, disorders, conditions, syndromes, and the like,being treated, prevented or promoted. Non-limiting examples of suchcarriers include liposomes, microsponges, microspheres, or microcapsulesof natural and synthetic polymers and the like. Liposomes may be formedfrom a variety of phospholipids such as cholesterol, stearylamines orphosphatidylcholines.

The progressive myocardial injury-preventing compositions of thedescribed invention may be administered parenterally in the form of asterile injectable aqueous or oleaginous suspension. The term“parenteral” or “parenterally” as used herein refers to introductioninto the body by way of an injection (i.e., administration byinjection), including, but not limited to, infusion techniques. In someembodiments, the progressive myocardial injury-preventing composition ofthe described invention comprising a chemotactic hematopoietic stem cellproduct is delivered to the subject by means of a balloon catheteradapted for delivery of the fluid compositions (i.e., compositionscapable of flow) into a selected anatomical structure.

The sterile progressive myocardial injury-preventing composition of thedescribed invention may be a sterile solution or suspension in anontoxic parenterally acceptable diluent or solvent. A solutiongenerally is considered as a homogeneous mixture of two or moresubstances; it is frequently, though not necessarily, a liquid. In asolution, the molecules of the solute (or dissolved substance) areuniformly distributed among those of the solvent. A suspension is adispersion (mixture) in which a finely-divided species is combined withanother species, with the former being so finely divided and mixed thatit does not rapidly settle out. In everyday life, the most commonsuspensions are those of solids in liquid water. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride (saline) solution. In some embodiments,hypertonic solutions are employed. In addition, sterile, fixed oilsconventionally are employed as a solvent or suspending medium. Forparenteral application, suitable vehicles consist of solutions, e.g.,oily or aqueous solutions, as well as suspensions, emulsions, orimplants. Aqueous suspensions may contain substances, which increase theviscosity of the suspension and include, for example, sodiumcarboxymethyl cellulose, sorbitol and/or dextran.

Additional progressive myocardial injury-preventing compositions of thedescribed invention readily may be prepared using technology, which isknown in the art, such as described in Remington's PharmaceuticalSciences, 18th or 19th editions, published by the Mack PublishingCompany of Easton, Pa., which is incorporated herein by reference.

As used herein the terms “therapeutically effective”, “myocardial injurypreventing amount”, “vascular insufficiency repairing amount”, “adverseconsequence preventing amount”, adverse consequence-reversing amount”,or “pharmaceutically effective amount” refer to the amount of thecompositions of the invention that result in a therapeutic or beneficialeffect following its administration to a subject. The vascularinsufficiency repairing, myocardial injury repairing, therapeutic,adverse consequence reversing or pharmaceutical effect may be curing,minimizing, preventing or ameliorating a disease or disorder, or mayhave any other vascular insufficiency-repairing, myocardialinjury-repairing, adverse consequence reversing, or pharmaceuticalbeneficial effect. The concentration of the substance is selected so asto exert its vascular insufficiency-repairing, myocardialinjury-repairing, adverse consequence reversing, therapeutic, orpharmaceutical effect, but low enough to avoid significant side effectswithin the scope and sound judgment of the physician. The effectiveamount of the composition may vary with the age and physical conditionof the biological subject being treated, the severity of the condition,the duration of the treatment, the nature of concurrent therapy, thetiming of the infusion, the specific compound, composition or otheractive ingredient employed, the particular carrier utilized, and likefactors.

A skilled artisan may determine a pharmaceutically effective amount ofthe inventive compositions by determining the dose in a dosage unit(meaning unit of use) that elicits a given intensity of effect,hereinafter referred to as the “unit dose.” The term “dose-intensityrelationship” refers to the manner in which the intensity of effect inan individual recipient relates to dose. The intensity of effectgenerally designated is 50% of maximum intensity. The corresponding doseis called the 50% effective dose or individual ED50. The use of the term“individual” distinguishes the ED50 based on the intensity of effect asused herein from the median effective dose, also abbreviated ED50,determined from frequency of response data in a population. “Efficacy”as used herein refers to the property of the compositions of thedescribed invention to achieve the desired response, and “maximumefficacy” refers to the maximum achievable effect. The amount of thechemotactic hematopoietic stem cell product in the pharmaceuticalcompositions of the described invention that will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and may be determined by standardclinical techniques. (See, for example, Goodman and Gilman's THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, Joel G. Harman, Lee E. Limbird,Eds.; McGraw Hill, New York, 2001; THE PHYSICIAN′S DESK REFERENCE,Medical Economics Company, Inc., Oradell, N.J., 1995; and DRUG FACTS ANDCOMPARISONS, FACTS AND COMPARISONS, INC., St. Louis, Mo., 1993), each ofwhich is incorporated by reference herein. The precise dose to beemployed in the formulations of the described invention also will dependon the route of administration and the seriousness of the disease ordisorder, and should be decided according to the judgment of thepractitioner and each subject's circumstances.

According to another embodiment, the pharmaceutical compositionsaccording to the described invention contain a minimum number of CD34+hematopoietic stem cells having a subpopulation of at least 0.5×10⁶CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotacticactivity per dosage unit for parenteral administration at thephysician's discretion. According to another embodiment, it isenvisioned that subjects can benefit from multiple administrations ofthe pharmaceutical compositions according to the described inventioncomprising a minimum number of CD34+ hematopoietic stem cells having asubpopulation of at least 0.5×10⁶ CD34+ cells expressing CXCR-4 andhaving CXCR-4 mediated chemotactic activity.

In another aspect of the described invention, the progressive myocardialinjury-preventing pharmaceutical compositions of the described inventionmay further include one or more compatible active ingredients, which areaimed at providing the progressive myocardial injury-preventingcomposition with another pharmaceutical effect in addition to thatprovided by the sterile chemotactic hematopoietic stem cell product ofthe described invention. “Compatible” as used herein means that theactive ingredients of such a composition are capable of being combinedwith each other in such a manner so that there is no interaction thatwould substantially reduce the efficacy of each active ingredient or thecomposition under ordinary use conditions. In some embodiments, thecombination therapy comprises administering to a subject in need thereofa progressive myocardial injury-preventing pharmaceutical compositioncomprising a sterile chemotactic hematopoietic stem cell product of thedescribed invention combined with an agent selected from the groupconsisting of an angiotensin converting enzyme (ACE) inhibitor, abeta-blocker, a diuretic, an anti-arrhythmic agent, a hematopoietic stemcell mobilizing agent, a tyrosine kinase receptor agonist, ananti-anginal agent, a vasoactive agent or inotrope, an anticoagulantagent, a fibrinolytic agent, and a hypercholesterolemic agent. Accordingto some embodiments, the tyrosine kinase receptor agonist isneuregulin 1. According to some embodiments, the neuregulin 1 is arecombinant protein. According to some embodiments, the hematopoieticstem cell mobilizing agent is a colony stimulating factor. According tosome such embodiments, the hematopoietic stem cell mobilizing agentcomprises G-CSF, GM-CSF, or a pharmaceutically acceptable analog orderivative thereof. According to some embodiments, the hematopoieticstem cell mobilizing agent is a recombinant analog or derivative of acolony stimulating factor. According to some embodiments, thehematopoietic stem cell mobilizing agent is filgrastim.

In some embodiments, the composition of the described invention furthercomprises about 0.5% to about 5% albumin. In some embodiments, theminimum amount of albumin is about 0.5% expressed as ml/100 cc volume ofthe composition. In some embodiments, the minimum amount of albumin isabout 0.75% expressed as ml/100 cc volume of the composition. In someembodiments, the minimum amount of albumin is about 1.0% expressed asml/100 cc volume of the composition. In some embodiments, the minimumamount of albumin is about 1.25% expressed as ml/100 cc volume of thecomposition. In some embodiments, the minimum amount of albumin is about1.5% expressed as ml/100 cc volume of the composition. In someembodiments, the minimum amount of albumin is about 1.75% expressed asml/100 cc volume of the composition. In some embodiments, the minimumamount of albumin is about 2.0% expressed as ml/100 cc volume of thecomposition. In some embodiments, the minimum amount of albumin is about2.5% expressed as ml/100 cc volume of the composition. In someembodiments, the minimum amount of albumin is about 2.75% expressed asml/100 cc volume of the composition. In some embodiments, the minimumamount of albumin is about 3.0% expressed as ml/100 cc volume of thecomposition. In some embodiments, the minimum amount of albumin is about3.5% expressed as ml/100 cc volume of the composition. In someembodiments, the minimum amount of albumin is about 4.0% expressed asml/100 cc volume of the composition. In some embodiments, the minimumamount of albumin is about 4.5% expressed as ml/100 cc volume of thecomposition. In some embodiments, the minimum amount of albumin is about5.0% expressed as ml/100 cc volume of the composition.

In some embodiments, the maximum amount of albumin in the compositionsof the described invention is about 5.0% expressed as ml/100 cc volumeof the composition. In some embodiments, the maximum amount of albuminin the compositions of the described invention is about 4.75% expressedas ml/100 cc volume of the composition. In some embodiments, the maximumamount of albumin in the compositions of the described invention isabout 4.5% expressed as ml/100 cc volume of the composition. In someembodiments, the maximum amount of albumin in the compositions of thedescribed invention is about 4.0% expressed as ml/100 cc volume of thecomposition. In some embodiments, the maximum amount of albumin in thecompositions of the described invention is about 4.25% expressed asml/100 cc volume of the composition. In some embodiments, the maximumamount of albumin in the compositions of the described invention isabout 4.0% expressed as ml/100 cc volume of the composition. In someembodiments, the maximum amount of albumin in the compositions of thedescribed invention is about 3.75% expressed as ml/100 cc volume of thecomposition. In some embodiments, the maximum amount of albumin in thecompositions of the described invention is about 3.5% expressed asml/100 cc volume of the composition. In some embodiments, the maximumamount of albumin in the compositions of the described invention isabout 3.25% expressed as ml/100 cc volume of the composition. In someembodiments, the maximum amount of albumin in the compositions of thedescribed invention is about 3.0% expressed as ml/100 cc volume of thecomposition. In some embodiments, the maximum amount of albumin in thecompositions of the described invention is about 2.75% expressed asml/100 cc volume of the composition. In some embodiments, the maximumamount of albumin in the compositions of the described invention isabout 2.0% expressed as ml/100 cc volume of the composition. In someembodiments, the maximum amount of albumin in the compositions of thedescribed invention is about 1.75% expressed as ml/100 cc volume of thecomposition. In some embodiments, the maximum amount of albumin in thecompositions of the described invention is about 1.5% expressed asml/100 cc volume of the composition. In some embodiments, the maximumamount of albumin in the compositions of the described invention isabout 1.25% expressed as ml/100 cc volume of the composition. In someembodiments, the maximum amount of albumin in the compositions of thedescribed invention is about 1% expressed as ml/100 cc volume of thecomposition. In some embodiments, the albumin is human albumin. In someembodiments the albumin is recombinant human albumin.

Methods of the Described Invention

In another aspect, the described invention provides a method ofpreparing a progressive myocardial injury-preventing pharmaceuticalcomposition comprising a sterile chemotactic hematopoietic stem cellproduct for treating a subject in need thereof. The method comprises thesteps of

(1) acquiring a sterile nonexpanded, isolated population of autologousmononuclear cells comprising CD34+ cells, which further contain asubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity from the subject under sterile conditions by achemotactic cell acquisition process;

(2) optionally freezing at least one aliquot of the nonexpanded,isolated population of autologous mononuclear cells of step (1) at −86°C. and cryostoring the at least one aliquot in the vapor phase of aliquid nitrogen freezer; and thawing the at least one aliquot of step(2) when needed;

(3) sterilely purifying the CD34+ cells from the sterile nonexpanded,isolated population of autologous mononuclear cells comprising CD34+cells of (1) or (2) so as to yield a chemotactic hematopoietic stem cellproduct comprising the nonexpanded, isolated population of autologousmononuclear cells enriched for CD34+ cells, which further contain asubpopulation of potent CD34+ cells that express CXCR-4 and that haveCXCR-4-mediated chemotactic activity;

(4) sterilely formulating the sterile chemotactic hematopoietic stemcell product to form a sterile pharmaceutical composition;

(5) confirming sterility of the pharmaceutical composition;

(6) releasing the sterile pharmaceutical composition as eligible forinfusion into the subject;

(7) loading a therapeutically effective amount of the pharmaceuticalcomposition into a chemotactic hematopoietic stem cell product deliveryapparatus; and

(8) optionally transporting the delivery apparatus containing thetherapeutically effective amount of the sterile pharmaceuticalcomposition comprising the sterile chemotactic hematopoietic stem cellproduct to a cardiac catheterization facility for infusion into thesubject.

According to some embodiments, the nonexpanded, isolated population ofautologous mononuclear cells can be acquired from the subject at anytime. According to some embodiments, the nonexpanded, isolatedpopulation of autologous mononuclear cells is acquired early after anAMI. According to some such embodiments, the nonexpanded, isolatedpopulation of autologous mononuclear cells is acquired 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days ormore after the occurrence of an AMI. According to some embodiments, thenonexpanded, isolated population of autologous mononuclear cells isacquired late after the occurrence of an AMI. According to some suchembodiments, the nonexpanded, isolated population of autologousmononuclear cells is acquired at least 15 days, at least 16 days, atleast 17 days, at least 18 days, at least 19 days, at least 20 days, atleast 21 days, at least 22 days, at least 23 days, at least 24 days, atleast 25 days, at least 26 days, at least 27 days, at least 28 days, atleast 29 days, at least 30 days, at least 60 days, at least 90 days, atleast 120 days, at least 150 days, at least 180 days or more after theoccurrence of the AMI. According to some embodiments, the nonexpanded,isolated population of autologous mononuclear cells is acquired at least1 month, at least 2 months, at least 3 months, at least 4 months, atleast 5 months, at least 6 months, at least 7 months, at least 8 months,at least 9 months, at least 10 months, at least 11 months, at least 12months, at least 16 months, at least 24 months, at least 30 months, atleast 36 months, at least 42 months, at least 48 months, at least 54months, at least 60 months, at least 66 months, at least 72 months, atleast 78 months, at least 84 months, at least 90 months, at least 96months, at least 102 months, at least 108 months, at least 114 months,at least 120 months, at least 126 months, at least 132 months, at least138 months, at least 144 months, at least 150 months, at least 156months, at least 162 months, at least 168 months, at least 174 months,at least 180 months, at least 186 months, at least 192 months, at least198 months, at least 204 months, at least 210 months, at least 216months, at least 222 months, at least 228 months, at least 234 months,at least 240 months or more after occurrence of an AMI. According tosome embodiments, the nonexpanded, isolated population of autologousmononuclear cells is acquired at least at least 3 years, 4 years, 5years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years,13 years, 14 years, 15 years, 16 years 17 years, 18 years, 19 years, 20years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34years, 35 years, 36 years 37 years, 38 years, 39 years, 40 years or moreafter occurrence of an AMI.

According to one embodiment, step (3) is initiated within about 12 hoursto about 24 hours of completion of acquiring step (1). According to someembodiments, releasing step (6) proceeds only if the sterile formulatedcell product is to be infused into the subject within about 48 hours toabout 72 hours of completion of acquiring step (1). According to anotherembodiment, step (3) is initiated within about 12 hours to about 24hours of completion of acquiring step (1), and releasing step (6)proceeds only if the sterile formulated cell product is to be infusedinto the subject within about 48 hours to about 72 hours of completionof acquiring step (1).

According to some embodiments, releasing step (6) proceeds only if thesterile formulated cell product is to be infused into the subject withinabout 48 hours to about 72 hours of thawing of the at least one frozenaliquot of optional step (2). According to another embodiment, step (3)is initiated within about 12 hours to about 24 hours of thawing of theat least one frozen aliquot of optional step (2), and releasing step (6)proceeds only if the sterile formulated cell product is to be infusedinto the subject within about 48 hours to about 72 hours of thawing ofthe at least one frozen aliquot of optional step (2).

According to some embodiments, a frozen aliquot of step (2) is thawed atleast 10 days, at least 11 days, at least 12 days, at least 13 days, atleast 14 days, at least 15 days, at least 16 days, at least 17 days, atleast 18 days, at least 19 days, at least 20 days, at least 21 days, atleast 22 days, at least 23 days, at least 24 days, at least 25 days, atleast 26 days, at least 27 days, at least 28 days, at least 29 days, atleast 30 days, at least 60 days, at least 90 days, at least 120 days, atleast 150 days, or at least 180 days, from the date the nonexpanded,isolated population of autologous mononuclear cells is acquired from thesubject in step (1). According to some embodiments, the frozen aliquotof step (4) is thawed at least 1 month, at least 2 months, at least 3months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, at least 12 months, at least 16 months, atleast 24 months, at least 30 months, at least 36 months, at least 42months, at least 48 months, at least 54 months, at least 60 months, atleast 66 months, at least 72 months, at least 78 months, at least 84months, at least 90 months, at least 96 months, at least 102 months, atleast 108 months, at least 114 months, at least 120 months, at least 126months, at least 132 months, at least 138 months, at least 144 months,at least 150 months, at least 156 months, at least 162 months, at least168 months, at least 174 months, at least 180 months, at least 186months, at least 192 months, at least 198 months, at least 204 months,at least 210 months, at least 216 months, at least 222 months, at least228 months, at least 234 months or at least 240 months from the date thenonexpanded, isolated population of autologous mononuclear cells isacquired from the subject in step (1). According to some embodiments,the frozen aliquot of step (2) is thawed at least 3 years, 4 years, 5years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years,13 years, 14 years, 15 years, 16 years 17 years, 18 years, 19 years, 20years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34years, 35 years, 36 years 37 years, 38 years, 39 years, 40 years or morefrom the date the nonexpanded, isolated population of autologousmononuclear cells is acquired from the subject in step (1).

According to such embodiments, the chemotactic hematopoietic stem cellproduct produced from the frozen aliquot is further characterized ashaving the following properties for at least 24 hours following thawingwhen tested in vitro after passage through a catheter: (1) retains atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70,%, at least 80%, at least 90%, or 100% of theCXCR-4-mediated activity of the of the subpopulation of potentCD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity; (2)at least 70% of the cells are CD34+ cells; (3) is at least 70% viable;and (4) is able to form hematopoietic colonies in vitro.

According to another embodiment, step (5), i.e., the step of assessingsterility of the pharmaceutical composition, further comprises the stepsof (i) centrifuging the sterile chemotactic hematopoietic stem cellproduct comprising potent CD34+/CXCR-4+ cells to form a cell pellet anda supernatant, the cell pellet comprising the potent CD34+/CXCR-4+cells; (ii) sterilely removing the supernatant without disturbing thecell pellet; and (iii) analyzing whether the supernatant is contaminatedby a microbe thereby determining the sterility of the cell pellet.

According to one embodiment, in step (1), the chemotactic cellacquisition process is a mini-bone marrow harvest technique used toacquire the nonexpanded isolated population of autologous mononuclearcells comprising potent CD34+/CXCR-4+ cells from the bone marrow of thesubject under sterile conditions. For the bone marrow harvest technique,step (1) of the method further comprises the steps: (i) preloadingharvesting syringes with heparin prior to harvesting bone marrow from asubject; (ii) aspirating the bone marrow from a left posterior iliaccrest and a right posterior iliac crest of the subject using theharvesting syringes and a mini-bone marrow harvest technique to formharvested bone marrow; and (iii) infusing the harvested bone marrow intoa collecting bag. In one embodiment, the harvesting syringes in step (i)and the collecting bag in step (iii) contain a preservative freeheparinized solution comprising 0.9% normal saline. The finalconcentration of heparin in the heparinized saline solution is about 20units per ml to about 25 units per ml.

Optionally, according to one embodiment of the method, the harvestedbone marrow is transported to a processing facility different from thefacility from which the bone marrow was harvested. According to oneembodiment, the method for transporting the harvested bone marrow to theprocessing facility comprises the steps (a) placing the harvested bonemarrow in a collection bag; (b) placing the collection bag in asecondary bag; (c) placing the secondary bag containing the collectionbag in a shipping container comprising an interior compartmentcontaining frozen wet ice and at least one sheet of bubble wrap; (d)affixing a temperature tag monitor to the interior compartment of theshipping container; (e) sealing the shipping container; and (f) shippingthe shipping container to the processing facility.

In another aspect, the described invention provides a method fortreating or preventing progressive myocardial injury due to a vascularinsufficiency that occurs early or late. The method comprising thesteps: (a) evaluating whether the subject qualifies for therapy with thepharmaceutical composition of the described invention; (b) preparing thepharmaceutical composition comprising a chemotactic hematopoietic stemcell product; (c) loading the pharmaceutical composition into achemotactic hematopoietic stem cell product delivery apparatus; (d)delivering a therapeutically effective amount of the pharmaceuticalcomposition to the subject; and (e) monitoring the subject's cardiacfunction. According to one embodiment, in step (d) the therapeuticallyeffective amount of the pharmaceutical composition is delivered to thesubject intravascularly (meaning inside a blood vessel). According toanother embodiment, the vascular insufficiency that occurs early or lateis an ischemia. According to some such embodiments, the ischemia is amyocardial ischemia. According to some such embodiments, the ischemia isa transient myocardial ischemia. According to some such embodiments, theischemia is a chronic myocardial ischemia. According to some suchembodiments, the ischemia is a peri-infarct border zone ischemia.According to one embodiment, the vascular insufficiency that occursearly or late is a vascular insufficiency after an acute myocardialinfarction resulting from underlying disease. According to some suchembodiments, the progressive myocardial injury is heart failure.

According to one embodiment of the described invention, the subject inneed thereof is a revascularized myocardial infarction patient. The term“revascularized” as used in this embodiment refers to the successfulplacement of a stent. Clinical evaluations, for example, of coronaryinsufficiency using non-laboratory tests, cardiac catheterization,measurement of inflammatory cytokines, and measurement of cardiacbiomarkers may be used to determine the appropriate time to administerthe pharmaceutical compositions in accordance with the methods of thedescribed invention. According to n some embodiments, detection of peakinflammatory cytokine cascade production enables the administration tobe tailored to the therapeutic window most crucial for the particularsubject. According to some embodiments, peak inflammatory cytokinecascade production is determined by measuring the levels of theappropriate cytokine(s) in the plasma and or urine. According to otherembodiments, the level(s) of the appropriate cytokine(s) is/are measuredimmunochemically, for example, by a sandwich enzyme immunoassay, byenzyme-linked immunosorbent assays (ELISA) or by multiplex bead kits.

According to some embodiments, the composition is administered at afirst infusion date. According to one embodiment, the first infusiondate is a time after an inflammatory cytokine cascade production peaks.According to some embodiments, the first infusion date at which thecomposition is administered to a revascularized myocardial infarctionpatient is about 5 days to about 14 days post-infarction. In someembodiments, the minimum first infusion date in which to administer thecomposition to a revascularized myocardial infarction patient is about5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days post-infarction. According tosome embodiments, the maximum first infusion date in which to administerthe composition to a revascularized myocardial infarction patient isabout 14, 12, 11, 10, 9, 8, 7, 6, or 5 days post-infarction.

According to some embodiments, the composition is administered multipletimes, or as needed in the judgment of the treating physician. Accordingto one such embodiment, the composition is administered at the firstinfusion date, and optionally at a second infusion date, a thirdinfusion date, a fourth infusion date, a fifth infusion date, a sixthinfusion date, a seventh infusion date, an eighth infusion date, a ninthinfusion date, a tenth infusion date, and so on.

According to some embodiments, the first infusion date at which thecomposition is administered to a revascularized subject suffering from avascular insufficiency that occurs early or late after a myocardialinfarction resulting from underlying disease comprises a specific timeinterval defined by a first time and a second time, wherein the firsttime is after peak inflammatory cytokine cascade production in theinfarcted area and the second time is before myocardial scar formationin the infarcted area.

According to some embodiments, the first infusion date is at least aboutone day, at least about two days, at least about three days, at leastabout four days, at least about five days, at least about six days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 15 days, atleast about 16 days, at least about 17 days, at least about 18 days, atleast about 19 days, at least about 20 days, at least about 21 days, atleast about 22 days, at least about 23 days, at least about 24 days, atleast about 25 days, at least about 26 days, at least about 27 days, atleast about 28 days, at least about 29 days, at least about 30 days ormore after occurrence of an AMI. According to some embodiments, thesecond infusion date is at least about 1 month, at least about 2 months,at least about 3 months, at least about 4 months, at least about 5months, at least about 6 months, at least about 7 months, at least about8 months, at least about 9 months, at least about 10 months, at leastabout 11 months, at least about 12 months, at least about 13 months, atleast about 14 months, at least about 15 months, at least about 16months, at least about 17 months, at least about 18 months, at leastabout 19 months, at least about 20 months, at least about 21 months, atleast about 22 months, at least about 23 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, at least about 72months, at least about 78 months, at least about 84 months, at leastabout 90 months, at least about 96 months, at least about 102 months, atleast about 108 months, at least about 114 months, at least about 120months, at least about 126 months, at least about 132 months, at leastabout 138 months, at least about 144 months, at least about 150 months,at least about 156 months, at least about 162 months, at least about 168months, at least about 174 months, at least about 180 months, at leastabout 186 months, at least about 192 months, at least about 198 months,at least about 204 months, at least about 210 months, at least about 216months, at least about 222 months, at least about 228 months, at leastabout 234 months, at least about 240 months or more after occurrence ofan AML According to some embodiments, the first infusion date is atleast 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years 17years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31years, 32 years, 33 years, 34 years, 35 years, 36 years 37 years, 38years, 39 years, 40 years or more after occurrence of an AMI.

According to some embodiments, the third infusion date is at least aboutone day, at least about two days, at least about three days, at leastabout four days, at least about five days, at least about six days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 15 days, atleast about 16 days, at least about 17 days, at least about 18 days, atleast about 19 days, at least about 20 days, at least about 21 days, atleast about 22 days, at least about 23 days, at least about 24 days, atleast about 25 days, at least about 26 days, at least about 27 days, atleast about 28 days, at least about 29 days, at least about 30 days ormore after occurrence of an AMI. According to some embodiments, thethird infusion date is at least about 1 month, at least about 2 months,at least about 3 months, at least about 4 months, at least about 5months, at least about 6 months, at least about 7 months, at least about8 months, at least about 9 months, at least about 10 months, at leastabout 11 months, at least about 12 months, at least about 13 months, atleast about 14 months, at least about 15 months, at least about 16months, at least about 17 months, at least about 18 months, at leastabout 19 months, at least about 20 months, at least about 21 months, atleast about 22 months, at least about 23 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, at least about 72months, at least about 78 months, at least about 84 months, at leastabout 90 months, at least about 96 months, at least about 102 months, atleast about 108 months, at least about 114 months, at least about 120months, at least about 126 months, at least about 132 months, at leastabout 138 months, at least about 144 months, at least about 150 months,at least about 156 months, at least about 162 months, at least about 168months, at least about 174 months, at least about 180 months, at leastabout 186 months, at least about 192 months, at least about 198 months,at least about 204 months, at least about 210 months, at least about 216months, at least about 222 months, at least about 228 months, at leastabout 234 months, at least about 240 months after occurrence of an AMI.According to some such embodiments, the third infusion date is at least3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years,11 years, 12 years, 13 years, 14 years, 15 years, 16 years 17 years, 18years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32years, 33 years, 34 years, 35 years, 36 years 37 years, 38 years, 39years, 40 years or more after occurrence of an AMI.

According to some embodiments, the fourth infusion date is at leastabout one day, at least about two days, at least about three days, atleast about four days, at least about five days, at least about sixdays, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 16 days, at least about 17 days, at least about 18days, at least about 19 days, at least about 20 days, at least about 21days, at least about 22 days, at least about 23 days, at least about 24days, at least about 25 days, at least about 26 days, at least about 27days, at least about 28 days, at least about 29 days, at least about 30days or more after occurrence of an AMI. According to some embodiments,the fourth infusion date is at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months, at least about 12 months, at least about 13months, at least about 14 months, at least about 15 months, at leastabout 16 months, at least about 17 months, at least about 18 months, atleast about 19 months, at least about 20 months, at least about 21months, at least about 22 months, at least about 23 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, at least about 48 months, at least about 54months, at least about 60 months, at least about 66 months, at leastabout 72 months, at least about 78 months, at least about 84 months, atleast about 90 months, at least about 96 months, at least about 102months, at least about 108 months, at least about 114 months, at leastabout 120 months, at least about 126 months, at least about 132 months,at least about 138 months, at least about 144 months, at least about 150months, at least about 156 months, at least about 162 months, at leastabout 168 months, at least about 174 months, at least about 180 months,at least about 186 months, at least about 192 months, at least about 198months, at least about 204 months, at least about 210 months, at leastabout 216 months, at least about 222 months, at least about 228 months,at least about 234 months, at least about 240 months or more afteroccurrence of an AMI. According to some such embodiments, the thirdinfusion date is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15years, 16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36years 37 years, 38 years, 39 years, 40 years or more after occurrence ofan AMI.

According to some embodiments, the fifth infusion date is at least aboutone day, at least about two days, at least about three days, at leastabout four days, at least about five days, at least about six days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 15 days, atleast about 16 days, at least about 17 days, at least about 18 days, atleast about 19 days, at least about 20 days, at least about 21 days, atleast about 22 days, at least about 23 days, at least about 24 days, atleast about 25 days, at least about 26 days, at least about 27 days, atleast about 28 days, at least about 29 days, at least about 30 days ormore after occurrence of an AMI. According to some embodiments, thefifth infusion date is at least about 1 month, at least about 2 months,at least about 3 months, at least about 4 months, at least about 5months, at least about 6 months, at least about 7 months, at least about8 months, at least about 9 months, at least about 10 months, at leastabout 11 months, at least about 12 months, at least about 13 months, atleast about 14 months, at least about 15 months, at least about 16months, at least about 17 months, at least about 18 months, at leastabout 19 months, at least about 20 months, at least about 21 months, atleast about 22 months, at least about 23 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, at least about 72months, at least about 78 months, at least about 84 months, at leastabout 90 months, at least about 96 months, at least about 102 months, atleast about 108 months, at least about 114 months, at least about 120months, at least about 126 months, at least about 132 months, at leastabout 138 months, at least about 144 months, at least about 150 months,at least about 156 months, at least about 162 months, at least about 168months, at least about 174 months, at least about 180 months, at leastabout 186 months, at least about 192 months, at least about 198 months,at least about 204 months, at least about 210 months, at least about 216months, at least about 222 months, at least about 228 months, at leastabout 234 months, at least about 240 months or more after occurrence ofan AMI. According to some embodiments, the first infusion date is atleast 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years 17years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31years, 32 years, 33 years, 34 years, 35 years, 36 years 37 years, 38years, 39 years, 40 years or more after occurrence of an AMI.

According to some embodiments, the sixth infusion date is at least aboutone day, at least about two days, at least about three days, at leastabout four days, at least about five days, at least about six days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 15 days, atleast about 16 days, at least about 17 days, at least about 18 days, atleast about 19 days, at least about 20 days, at least about 21 days, atleast about 22 days, at least about 23 days, at least about 24 days, atleast about 25 days, at least about 26 days, at least about 27 days, atleast about 28 days, at least about 29 days, at least about 30 days ormore after occurrence of an AMI. According to some embodiments, thesixth infusion date is at least about 1 month, at least about 2 months,at least about 3 months, at least about 4 months, at least about 5months, at least about 6 months, at least about 7 months, at least about8 months, at least about 9 months, at least about 10 months, at leastabout 11 months, at least about 12 months, at least about 13 months, atleast about 14 months, at least about 15 months, at least about 16months, at least about 17 months, at least about 18 months, at leastabout 19 months, at least about 20 months, at least about 21 months, atleast about 22 months, at least about 23 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, at least about 72months, at least about 78 months, at least about 84 months, at leastabout 90 months, at least about 96 months, at least about 102 months, atleast about 108 months, at least about 114 months, at least about 120months, at least about 126 months, at least about 132 months, at leastabout 138 months, at least about 144 months, at least about 150 months,at least about 156 months, at least about 162 months, at least about 168months, at least about 174 months, at least about 180 months, at leastabout 186 months, at least about 192 months, at least about 198 months,at least about 204 months, at least about 210 months, at least about 216months, at least about 222 months, at least about 228 months, at leastabout 234 months, at least about 240 months or more after occurrence ofan AMI. According to some such embodiments, the third infusion date isat least 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years,10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years 17years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31years, 32 years, 33 years, 34 years, 35 years, 36 years 37 years, 38years, 39 years, 40 years or more after occurrence of an AMI.

According to some such embodiments, the seventh infusion date is atleast about one day, at least about two days, at least about three days,at least about four days, at least about five days, at least about sixdays, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 16 days, at least about 17 days, at least about 18days, at least about 19 days, at least about 20 days, at least about 21days, at least about 22 days, at least about 23 days, at least about 24days, at least about 25 days, at least about 26 days, at least about 27days, at least about 28 days, at least about 29 days, at least about 30days or more after occurrence of an AMI. According to some embodiments,the seventh infusion date is at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months, at least about 12 months, at least about 13months, at least about 14 months, at least about 15 months, at leastabout 16 months, at least about 17 months, at least about 18 months, atleast about 19 months, at least about 20 months, at least about 21months, at least about 22 months, at least about 23 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, at least about 48 months, at least about 54months, at least about 60 months, at least about 66 months, at leastabout 72 months, at least about 78 months, at least about 84 months, atleast about 90 months, at least about 96 months, at least about 102months, at least about 108 months, at least about 114 months, at leastabout 120 months, at least about 126 months, at least about 132 months,at least about 138 months, at least about 144 months, at least about 150months, at least about 156 months, at least about 162 months, at leastabout 168 months, at least about 174 months, at least about 180 months,at least about 186 months, at least about 192 months, at least about 198months, at least about 204 months, at least about 210 months, at leastabout 216 months, at least about 222 months, at least about 228 months,at least about 234 months, at least about 240 months or more afteroccurrence of an AMI. According to some such embodiments, the thirdinfusion date is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15years, 16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36years 37 years, 38 years, 39 years, 40 years or more after occurrence ofan AMI.

According to some such embodiments, the eighth infusion date is at leastabout one day, at least about two days, at least about three days, atleast about four days, at least about five days, at least about sixdays, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 16 days, at least about 17 days, at least about 18days, at least about 19 days, at least about 20 days, at least about 21days, at least about 22 days, at least about 23 days, at least about 24days, at least about 25 days, at least about 26 days, at least about 27days, at least about 28 days, at least about 29 days, at least about 30days or more after occurrence of an AMI. According to some embodiments,the eighth infusion date is at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months, at least about 12 months, at least about 13months, at least about 14 months, at least about 15 months, at leastabout 16 months, at least about 17 months, at least about 18 months, atleast about 19 months, at least about 20 months, at least about 21months, at least about 22 months, at least about 23 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, at least about 48 months, at least about 54months, at least about 60 months, at least about 66 months, at leastabout 72 months, at least about 78 months, at least about 84 months, atleast about 90 months, at least about 96 months, at least about 102months, at least about 108 months, at least about 114 months, at leastabout 120 months, at least about 126 months, at least about 132 months,at least about 138 months, at least about 144 months, at least about 150months, at least about 156 months, at least about 162 months, at leastabout 168 months, at least about 174 months, at least about 180 months,at least about 186 months, at least about 192 months, at least about 198months, at least about 204 months, at least about 210 months, at leastabout 216 months, at least about 222 months, at least about 228 months,at least about 234 months, at least about 240 months or more afteroccurrence of an AMI. According to some such embodiments, the thirdinfusion date is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15years, 16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36years 37 years, 38 years, 39 years, 40 years or more after occurrence ofan AMI.

According to some such embodiments, the ninth infusion date is at leastabout one day, at least about two days, at least about three days, atleast about four days, at least about five days, at least about sixdays, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 16 days, at least about 17 days, at least about 18days, at least about 19 days, at least about 20 days, at least about 21days, at least about 22 days, at least about 23 days, at least about 24days, at least about 25 days, at least about 26 days, at least about 27days, at least about 28 days, at least about 29 days, at least about 30days or more after occurrence of an AMI. According to some embodiments,the ninth infusion date is at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months, at least about 12 months, at least about 13months, at least about 14 months, at least about 15 months, at leastabout 16 months, at least about 17 months, at least about 18 months, atleast about 19 months, at least about 20 months, at least about 21months, at least about 22 months, at least about 23 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, at least about 48 months, at least about 54months, at least about 60 months, at least about 66 months, at leastabout 72 months, at least about 78 months, at least about 84 months, atleast about 90 months, at least about 96 months, at least about 102months, at least about 108 months, at least about 114 months, at leastabout 120 months, at least about 126 months, at least about 132 months,at least about 138 months, at least about 144 months, at least about 150months, at least about 156 months, at least about 162 months, at leastabout 168 months, at least about 174 months, at least about 180 months,at least about 186 months, at least about 192 months, at least about 198months, at least about 204 months, at least about 210 months, at leastabout 216 months, at least about 222 months, at least about 228 months,at least about 234 months, at least about 240 months or more afteroccurrence of an AMI. According to some such embodiments, the thirdinfusion date is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15years, 16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36years 37 years, 38 years, 39 years, 40 years or more after occurrence ofan AMI.

According to some such embodiments, the tenth infusion date is at leastabout one day, at least about two days, at least about three days, atleast about four days, at least about five days, at least about sixdays, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 16 days, at least about 17 days, at least about 18days, at least about 19 days, at least about 20 days, at least about 21days, at least about 22 days, at least about 23 days, at least about 24days, at least about 25 days, at least about 26 days, at least about 27days, at least about 28 days, at least about 29 days, at least about 30days or more after occurrence of an AMI. According to some embodiments,the tenth infusion date is at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months, at least about 12 months, at least about 13months, at least about 14 months, at least about 15 months, at leastabout 16 months, at least about 17 months, at least about 18 months, atleast about 19 months, at least about 20 months, at least about 21months, at least about 22 months, at least about 23 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, at least about 48 months, at least about 54months, at least about 60 months, at least about 66 months, at leastabout 72 months, at least about 78 months, at least about 84 months, atleast about 90 months, at least about 96 months, at least about 102months, at least about 108 months, at least about 114 months, at leastabout 120 months, at least about 126 months, at least about 132 months,at least about 138 months, at least about 144 months, at least about 150months, at least about 156 months, at least about 162 months, at leastabout 168 months, at least about 174 months, at least about 180 months,at least about 186 months, at least about 192 months, at least about 198months, at least about 204 months, at least about 210 months, at leastabout 216 months, at least about 222 months, at least about 228 months,at least about 234 months, at least about 240 months or more afteroccurrence of an AMI. According to some such embodiments, the thirdinfusion date is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15years, 16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36years 37 years, 38 years, 39 years, 40 years or more after occurrence ofan AMI, and so on.

According to some embodiments, the chemotactic hematopoietic stem cellproduct of the composition administered at the second, third, fourth,fifth, sixth, seventh, eighth, ninth and/or tenth infusion date isprepared from a frozen and thawed aliquot of a nonexpanded, isolatedpopulation of autologous mononuclear cells containing CD34+ cells.

According to some embodiments, the chemotactic hematopoietic stem cellproduct delivery apparatus used to deliver the pharmaceuticalcomposition of the described invention to a subject in need thereofcomprises an infusion syringe, a flushing syringe, a four-way stopcock,and a balloon catheter. In one embodiment, the intravascular deliverycomprises (a) an infusion device attached to a sterile four-way stopcockcontaining the pharmaceutical composition comprising the chemotactichematopoietic stem cell product; (b) a flushing device attached to thesterile four-way stopcock, the flushing device containing a flushingsolution, and (c) a catheter attached to the delivery apparatus by thesterile four-way stopcock. According to one embodiment, the infusiondevice is a syringe made of any suitable material. The body and handleof suitable four way stopcocks may be made of the same or a differentmaterial. Examples of suitable four-way stopcocks includes, withoutlimitation, a stopcock having a polycarbonate body/polycarbonate handle,a stopcock having a polyethylene body/polyethylene handle, a stopcockhaving a polycarbonate body/polyethylene handle, or a disposablestopcock. According to some embodiments, a device is further attached tothe stopcock to regulate the pressure exerted on the delivered solution.According to some embodiments, an integral flush device or syringe isattached to the stopcock. According to one embodiment, the catheter is aballoon catheter. The term “balloon catheter” refers to a type of “soft”thin flexible tube having an inflatable “balloon” at its tip, which isused during a catheterization procedure to enlarge a narrow opening orpassage within the body. The deflated balloon catheter is positioned,inflated to perform the necessary procedure, and deflated again to beremoved.

The viability and potential efficacy of the chemotactic hematopoieticstem cell product of the described invention comprising potentCD34+/CXCR-4+ cells depends on the cells maintaining their potency asthey pass through a catheter. The catheter used in the methods of thedescribed invention has an internal diameter of at least 0.36 mm. Anytype of catheter having an internal diameter of at least 0.36 mm may beeffective in delivering the pharmaceutical compositions of the describedinvention.

For example, a flow control catheter, which slows drainage of bloodthrough the coronary artery vasculature, allows the cells time totransit through the blood vessel wall and into the tissue.

In some embodiments, the catheter is a balloon catheter. For example,without limitation, the following balloon dilatation catheters availablefrom Cordis, Boston Scientific, Medtronic and Guidant having an internaldiameter of about 0.36 mm have been validated (see Table 1).

TABLE 1 Balloon catheter validated for infusion of selected CD34⁺ cellsthrough the IRA Lumen Name and Model Balloon Internal Manufacturer No.Dimensions Diameter Cordis Raptor OTW 579-130 15 mm × 3.0 mm  0.36 mm(0.14 in.) Boston Scientific OTW Maverick 15 mm × 3.0 mm  0.36 mm20620-1530 (0.14 in.) Medtronic OTW Sprinter SPR 15 mm × 3.0 mm  0.36 mm3015W (0.14 in.) Guidant Voyager OTW 15 mm × 3.0 mm  0.36 mm 1009443-15(0.14 in.)

In addition, catheters have been described having a fluid delivery portadjacent to the balloon such that the balloon may be inflated against avessel wall to isolate the delivery site from hemodynamics opposite theballoon from the port, which may be located distally of the balloon.Additionally, balloon catheters have been disclosed having lumens endingin side ports disposed proximally to the balloon catheter; these ballooncatheters generally may be referred to as “balloon/delivery” catheters,although particular references may use different descriptors. See, e.g.,U.S. Pat. No. 5,415,636 to Forman, incorporated by reference herein.

According to some embodiments, the method of treating or preventing aprogressive myocardial injury due to a vascular insufficiency thatoccurs early or late comprises administering the progressive myocardialinjury-preventing pharmaceutical composition via balloon catheterizationinto an artery at a first infusion date. In some embodiments, followingangioplasty, a delivery balloon catheter is inserted via a femoralartery into a desired coronary artery, such as the left anteriordescending coronary artery. Some medical conditions may require both aballoon catheter and a fluid delivery catheter to facilitate treatment.

According to some embodiments, a catheter is used to directly injectcells into the myocardium.

Treatment Regimens

According to another aspect, the described invention provides a regimenfor treating a progressive myocardial injury due to a vascularinsufficiency that occurs early or late, which comprises:

(a) first administering to the subject on a first infusion date a firststerile pharmaceutical composition parenterally through a catheter, thefirst sterile pharmaceutical composition of (a) comprising: (i) atherapeutically effective amount of a first sterile chemotactichematopoietic stem cell product, wherein the first chemotactichematopoietic stem cell product comprises a nonexpanded, isolatedpopulation of autologous mononuclear cells enriched for CD34+ cells,which further contain a subpopulation of potent CD34+/CXCR-4+ cells thathave CXCR-4-mediated chemotactic activity, wherein the therapeuticallyeffective amount of the first chemotactic hematopoietic stem cellproduct comprises at least 10×10⁶ CD34+ cells containing at least0.5×10⁶ potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediatedchemotactic activity; (ii) a stabilizing amount of serum, wherein thestabilizing amount of serum is greater than 20% (v/v), wherein thechemotactic hematopoietic stem cell product is further characterized ashaving the following properties for at least 24 hours followingacquisition of the chemotactic hematopoietic stem cell product whentested in vitro after passage through a catheter: (1) retains theCXCR-4-mediated activity of the subpopulation of potent CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity; (2) at least 70%of the cells are CD34+ cells; (3) is at least 70% viable; and (4) isable to form hematopoietic colonies in vitro;

(b) second, administering at a second infusion date a second sterilepharmaceutical composition comprising a therapeutic amount of asecondchemotactic hematopoietic stem cell product; wherein thetherapeutically effective amount of the second chemotactic hematopoieticstem cell product comprises at least 10×10⁶ CD34+ cells which furthercontain a subpopulation of at least 0.5×10⁶ potent CD34+ cellsexpressing CXCR-4 and having CXCR-4 mediated chemotactic activity; (ii)a stabilizing amount of serum, wherein the stabilizing amount of serumis greater than 20% (v/v), wherein the secondchemotactic hematopoieticstem cell product is further characterized as having the followingproperties for at least 24 hours when tested in vitro after passagethrough a catheter: (1) retains the CXCR-4-mediated activity of thesubpopulation of potent CD34-/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity; (2) at least 70% of the cells are CD34+ cells; (3)is at least 70% viable; and (4) is able to form hematopoietic coloniesin vitro; and

(c) third, optionally administering at a third infusion date a sterilepharmaceutical composition comprising a third chemotactic hematopoieticstem cell product comprising at least 10×10⁶ isolated CD34+ cells, whichfurther contain a subpopulation of at least 0.5×10⁶ potent CD34+ cellsexpressing CXCR-4 and having CXCR-4 mediated chemotactic activity; (ii)a stabilizing amount of serum, wherein the stabilizing amount of serumis greater than 20% (v/v), wherein the third chemotactic hematopoieticstem cell product is further characterized as having the followingproperties for at least 24 hours when tested in vitro after passagethrough a catheter: (1) retains the CXCR-4-mediated activity of thesubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity; (2) at least 70% of the cells are CD34+ cells; (3)is at least 70% viable; and (4) is able to form hematopoietic coloniesin vitro, such that the regimen improves at least one measure of cardiacfunction.

According to some embodiments, at least one aliquot of the sterilenonexpanded, isolated population of autologous mononuclear cellscomprising CD34+ cells, which further contain a subpopulation of potentCD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activityacquired from the subject under sterile conditions is frozen at −86° C.and cryostored at least one aliquot in the vapor phase of a liquidnitrogen freezer until needed. At that time, the at least one aliquot ofthe frozen nonexpanded, isolated population of autologous mononuclearcells containing CD34+ cells which further contain a subpopulation ofpotent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotacticactivity is thawed and enriched for CD34+ cells, which further contain asubpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediatedchemotactic activity. This frozen and thawed nonexpanded, isolatedpopulation of autologous mononuclear cells enriched for CD34+ cellswhich further contain a subpopulation of potent CD34+/CXCR-4+ cells thathave CXCR-4-mediated chemotactic activity constitutes a thawed sterilechemotactic hematopoietic stem cell product.

According to some embodiments, the thawed sterile chemotactichematopoietic stem cell product can be used in step (b), step (c), orsteps (b) and step (c) of the regimen.

The term “regimen” as used herein refers to a course or plan oftreatment to preserve or restore the health of a subject suffering froma progressive myocardial injury due to a vascular insufficiency thatoccurs early or late.

According to one embodiment of the regimen, the thawed sterilechemotactic hematopoietic stem cell product, when passed through thecatheter and tested in vitro, (i) is able to form hematopoieticcolonies; and (ii) retains at least 2% of the CXCR-4-mediated activityof the subpopulation of potent CD34+/CXCR-4+ cells that haveCXCR-4-mediated chemotactic activity, for at least 48 hours followingthawing of the cryostored nonexpanded, isolated population of autologousmononuclear cells comprising CD34+ cells, which further contain asubpopulation of potent CD34+/CXCR-4-+ cells that have CXCR-4-mediatedchemotactic activity. According to another embodiment, the thawedchemotactic hematopoietic stem cell product, when passed through thecatheter and tested in vitro, (i) is able to form hematopoieticcolonies; and (ii) retains at least 2% of the CXCR-4-mediated activityof the subpopulation of potent CD34+/CXCR-4+ cells that haveCXCR-4-mediated chemotactic activity for at least 72 hours followingthawing of nonexpanded, isolated population of autologous mononuclearcells comprising CD34+ cells, which further contain a subpopulation ofpotent CD34+/CXCR-4-+ cells that have CXCR-4-mediated chemotacticactivity.

According to another embodiment, the first infusion date of (a) is atleast is at least about one day, at least about two days, at least aboutthree days, at least about four days, at least about five days, at leastabout six days, at least about 7 days, at least about 8 days, at leastabout 9 days, at least about 10 days, at least about 11 days, at leastabout 12 days, at least about 13 days, at least about 14 days, at leastabout 15 days, at least about 16 days, at least about 17 days, at leastabout 18 days, at least about 19 days, at least about 20 days, at leastabout 21 days, at least about 22 days, at least about 23 days, at leastabout 24 days, at least about 25 days, at least about 26 days, at leastabout 27 days, at least about 28 days, at least about 29 days, at leastabout 30 days or more after occurrence of an AMI. According to someembodiments, the first infusion date of (a) is at least about 1 month,at least about 2 months, at least about 3 months, at least about 4months, at least about 5 months, at least about 6 months, at least about7 months, at least about 8 months, at least about 9 months, at leastabout 10 months, at least about 11 months, at least about 12 months, atleast about 13 months, at least about 14 months, at least about 15months, at least about 16 months, at least about 17 months, at leastabout 18 months, at least about 19 months, at least about 20 months, atleast about 21 months, at least about 22 months, at least about 23months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, at least about 48 months, atleast about 54 months, at least about 60 months, at least about 66months, at least about 72 months, at least about 78 months, at leastabout 84 months, at least about 90 months, at least about 96 months, atleast about 102 months, at least about 108 months, at least about 114months, at least about 120 months, at least about 126 months, at leastabout 132 months, at least about 138 months, at least about 144 months,at least about 150 months, at least about 156 months, at least about 162months, at least about 168 months, at least about 174 months, at leastabout 180 months, at least about 186 months, at least about 192 months,at least about 198 months, at least about 204 months, at least about 210months, at least about 216 months, at least about 222 months, at leastabout 228 months, at least about 234 months, at least about 240 monthsor more after occurrence of an AMI. According to some embodiments, thefirst infusion date is at least 3 years, 4 years, 5 years, 6 years, 7years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14years, 15 years, 16 years 17 years, 18 years, 19 years, 20 years, 21years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35years, 36 years 37 years, 38 years, 39 years, 40 years or more afteroccurrence of an AMI.

According to another embodiment, the second infusion date of (b) is atleast about is at least about one day, at least about two days, at leastabout three days, at least about four days, at least about five days, atleast about six days, at least about 7 days, at least about 8 days, atleast about 9 days, at least about 10 days, at least about 11 days, atleast about 12 days, at least about 13 days, at least about 14 days, atleast about 15 days, at least about 16 days, at least about 17 days, atleast about 18 days, at least about 19 days, at least about 20 days, atleast about 21 days, at least about 22 days, at least about 23 days, atleast about 24 days, at least about 25 days, at least about 26 days, atleast about 27 days, at least about 28 days, at least about 29 days, atleast about 30 days or more after occurrence of an AMI. According tosome embodiments, the third infusion date of (c) is at least about 1month, at least about 2 months, at least about 3 months, at least about4 months, at least about 5 months, at least about 6 months, at leastabout 7 months, at least about 8 months, at least about 9 months, atleast about 10 months, at least about 11 months, at least about 12months, at least about 13 months, at least about 14 months, at leastabout 15 months, at least about 16 months, at least about 17 months, atleast about 18 months, at least about 19 months, at least about 20months, at least about 21 months, at least about 22 months, at leastabout 23 months, at least about 24 months, at least about 30 months, atleast about 36 months, at least about 42 months, at least about 48months, at least about 54 months, at least about 60 months, at leastabout 66 months, at least about 72 months, at least about 78 months, atleast about 84 months, at least about 90 months, at least about 96months, at least about 102 months, at least about 108 months, at leastabout 114 months, at least about 120 months, at least about 126 months,at least about 132 months, at least about 138 months, at least about 144months, at least about 150 months, at least about 156 months, at leastabout 162 months, at least about 168 months, at least about 174 months,at least about 180 months, at least about 186 months, at least about 192months, at least about 198 months, at least about 204 months, at leastabout 210 months, at least about 216 months, at least about 222 months,at least about 228 months, at least about 234 months, at least about 240months or more after occurrence of an AMI. According to someembodiments, the first infusion date is at least 3 years, 4 years, 5years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years,13 years, 14 years, 15 years, 16 years 17 years, 18 years, 19 years, 20years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34years, 35 years, 36 years 37 years, 38 years, 39 years, 40 years or moreafter occurrence of an AMI.

According to another embodiment, the third infusion date of (c) is atleast about is at least about one day, at least about two days, at leastabout three days, at least about four days, at least about five days, atleast about six days, at least about 7 days, at least about 8 days, atleast about 9 days, at least about 10 days, at least about 11 days, atleast about 12 days, at least about 13 days, at least about 14 days, atleast about 15 days, at least about 16 days, at least about 17 days, atleast about 18 days, at least about 19 days, at least about 20 days, atleast about 21 days, at least about 22 days, at least about 23 days, atleast about 24 days, at least about 25 days, at least about 26 days, atleast about 27 days, at least about 28 days, at least about 29 days, orat least about 30 days after occurrence of an AMI. According to someembodiments, the third infusion date of (c) is at least about 1 month,at least about 2 months, at least about 3 months, at least about 4months, at least about 5 months, at least about 6 months, at least about7 months, at least about 8 months, at least about 9 months, at leastabout 10 months, at least about 11 months, at least about 12 months, atleast about 13 months, at least about 14 months, at least about 15months, at least about 16 months, at least about 17 months, at leastabout 18 months, at least about 19 months, at least about 20 months, atleast about 21 months, at least about 22 months, at least about 23months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, at least about 48 months, atleast about 54 months, at least about 60 months, at least about 66months, at least about 72 months, at least about 78 months, at leastabout 84 months, at least about 90 months, at least about 96 months, atleast about 102 months, at least about 108 months, at least about 114months, at least about 120 months, at least about 126 months, at leastabout 132 months, at least about 138 months, at least about 144 months,at least about 150 months, at least about 156 months, at least about 162months, at least about 168 months, at least about 174 months, at leastabout 180 months, at least about 186 months, at least about 192 months,at least about 198 months, at least about 204 months, at least about 210months, at least about 216 months, at least about 222 months, at leastabout 228 months, at least about 234 months, at least about 240 monthsor more after occurrence of an AMI. According to some embodiments, thefirst infusion date is at least 3 years, 4 years, 5 years, 6 years, 7years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14years, 15 years, 16 years 17 years, 18 years, 19 years, 20 years, 21years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35years, 36 years 37 years, 38 years, 39 years, 40 years or more afteroccurrence of an AMT.

According to another embodiment, the vascular insufficiency that occursearly or late is an ischemia. According to another embodiment, theischemia is a myocardial ischemia. According to another embodiment, theischemia is a transient ischemia. According to another embodiment, theischemia is a chronic myocardial ischemia. According to anotherembodiment, the ischemia is a peri-infarct border zone ischemia.According to another embodiment, the catheter is a flow controlcatheter. According to another embodiment, the catheter is a balloondilatation catheter. According to another embodiment, the catheter hasan internal diameter of at least about 0.36 mm. According to anotherembodiment, the composition is administered through the catheter intomyocardium. According to another embodiment, the composition isadministered through the catheter intravascularly. According to anotherembodiment, the pharmaceutical composition further includes at least onecompatible active agent. According to another embodiment, the activeagent is selected from the group consisting of an angiotensin convertingenzyme inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent,a hematopoietic stem cell mobilizing agent, a tyrosine kinase receptoragonist, an anti-anginal agent, a vasoactive agent, an anticoagulantagent, a fibrinolytic agent, and a hypercholesterolemic agent. Accordingto another embodiment, the tyrosine kinase receptor agonist is humanneuregulin 1. According to some embodiments, the hematopoietic stem cellmobilizing agent is a colony stimulating factor. According to some suchembodiments, the hematopoietic stem cell mobilizing agent comprisesG-CSF, GM-CSF, or a pharmaceutically acceptable analog or derivativethereof. According to some embodiments, the hematopoietic stem cellmobilizing agent is a recombinant analog or derivative of a colonystimulating factor. According to some embodiments, the hematopoieticstem cell mobilizing agent is filgrastim.

According to another embodiment, the vascular insufficiency that occursearly or late is a vascular insufficiency after an acute myocardialinfarction resulting from underlying disease. According to some suchembodiments, the first infusion date comprises a specific time intervaldefined by a first time and a second time, wherein the first time isafter peak inflammatory cytokine cascade production in the infarctedarea and the second time is before myocardial scar formation in theinfarcted area. According to another embodiment, in step (a), the firsttime of the first infusion date is at least about 5 dayspost-infarction. According to another embodiment, in step (a) the firsttime of the first infusion date is about 5 days post-infarction and thesecond time is about 14 days post-infarction. According to anotherembodiment, the regimen treats cardiomyocyte cell death in theperi-infarct border zone, relative to controls. According to anotherembodiment, the regimen treats hypoperfusion in the pen-infarct borderzone, relative to controls. According to another embodiment, the regimentreats myocardial hibernation in the pen-infarct border zone, relativeto controls. According to another embodiment, the regimen decreasesinfarct area relative to controls. According to another embodiment, theregimen decreases infarct mass, relative to controls. According toanother embodiment, the progressive myocardial injury is a progressivedecline in heart muscle function following the acute mycocardialinfarction. According to another embodiment, the progressive myocardialinjury is heart failure.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges also is encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the described invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

As used herein and in the appended claims, the singular forms “a”,“and”, and “the” include plural referents unless the context clearlydictates otherwise. All technical and scientific terms used herein havethe same meaning.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the described inventionis not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided may be differentfrom the actual publication dates which may need to be confirmedindependently.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Phase I Clinical Trial

Example 1 Selection of Eligible Subjects

Subjects/patients presenting with symptoms and clinical findingssuggestive of a myocardial infarction will receive emergency diagnosticand clinical management according to institutional guidelines. If atransmural (meaning through the wall) myocardial infarction isconfirmed, the time of first symptoms and the time of successful stentplacement will be recorded. Revascularized subjects will receiveappropriate medical management to reduce ventricular wall stressesaccording to institutional guidelines. The term “revascularized” as usedin this embodiment, refers to the successful placement of a stent.

All types of stents, including drug-eluting stents (e.g., paclitaxel orsirolimus) are acceptable for use in the revascularization of theinfarct related artery (“IRA”). Previous studies employing ballooncatheters to infuse cell products have reported no limits for referencevessel diameter for the placement of the stent. Since this study isdesigned to distribute the cell product into the IRA circulation, and inan attempt to limit the potential for damage to very small vessels, thedescribed invention requires that stents be placed prior to infusion ofthe chemotactic hematopoietic stem cell product of the describedinvention.

Stent-related drug effects occur predominantly at the site of contact ofthe stent with the vessel wall. Consequent to balloon dilatation, thereis limited blood flow across the stent during cell infusion, andtherefore no significant adverse drug-mediated effect on the CD34+ cellsin the chemotactic hematopoietic stem cell product is expected.Moreover, prior clinical studies have shown that by 96 hours afterdrug-eluting stent placement, whole blood levels of either paclitaxel orsirolimus are below the limits of detection. Therefore, tissue levels inthe myocardial sites to which the infused CD34+ cells expressing CXCR-4and having CXCR-4-mediated chemotactic activity are intended to migrateare expected to be inconsequential. See Sousa, J. et al., Circulation107: 2274-79, 2383-89 (2003).

During revascularization, a subject's cardiac function and perfusionwill be assessed by standard methods. Relevant measures of cardiacfunction include assessment of global and regional ejection fraction,ventricular volumes, resting and stress perfusion, segmented wallmotion, and, following a myocardial infarction, infarct size.

The term “diastole” refers to the normal postsystolic dilation of theheart cavities during which they fill with blood. The term “systole”refers to contraction of the heart, especially of the ventricles, bywhich the blood is driven through the aorta and pulmonary artery totraverse the systemic and pulmonary circulations, respectively.

The term “ejection fraction” (“EF”) refers to the percentage of bloodemptied from the ventricle during contraction More specifically, it isthe fraction of the end-diastolic volume that is ejected with each beat;that is, it is stroke volume (SV) divided by end-diastolic volume (EDV).The volume of blood within a ventricle immediately before a contractionis known as the end-diastolic volume, while the volume of blood left ina ventricle at the end of contraction is known as end-systolic volume.The difference between end-diastolic and end-systolic volumes is thestroke volume, the volume of blood ejected with each beat. In a healthy70-kg (154-lb) male, the SV is approximately 70 ml and the leftventricular EDV is 120 ml, giving an ejection fraction of 70/120, or0.58 (58%). An EF within the range of from 55-60% is considered normal.The ejection fraction of the right ventricle (“RVEF”) normally is equalto that of the left ventricle (“LVEF”) within narrow limits.

Other measures of cardiac function include assessment of the strokevolume index and velocity of circumferential fiber shortening. Strauer,et al., Circulation 106: 1913-18 (2002). Stroke volume (SV) is theamount of blood the left ventricle ejects in one beat, measured inmilliliters per beat (ml/beat). SV can be indexed to a patient's bodysize by dividing SV by Body Surface Area (BSA) to yield the Stroke Index(SI).

Assessment of repair of infarcted myocardium also has includedevaluation of peri-infarct region perfusion using thallium scintigraphy.Id. The term “perfusion” refers to the process of nutritive delivery ofarterial blood to a capillary bed in biological tissue. Perfusion (“F”)may be calculated using the formula F=(Pa−Pv)/R, where Pa is meanarterial pressure, Pv is mean venous pressure, and R is vascularresistance:

Magnetic resonance imaging (MR1) is a useful tool for assessing cardiacfunction and viability (infarct size) in this setting. See Yin, A, etal., Blood 90: 5002-5012 (1997).

The day after successful stenting, subjects will be assessed for studyeligibility and, if appropriate, will be offered informed consent toparticipate in the study. Subjects exhibiting symptoms for no more thanthree (3) days prior to successful stent placement will be assessed,prior to discharge, for study eligibility. Subjects found to meeteligibility criteria (see infra) will be offered informed consent toparticipate.

Consented subjects will have a study entry SPECT no sooner than 96 hoursafter stent placement. Subjects are eligible to proceed on study if theLVEF is less than or equal to 50% on echocardiography and a segmentalventricular wall abnormality is observed in the IRA. Eligible subjectsimmediately can complete baseline cardiac function and perfusionassessment.

Specifically, baseline cardiac function includes:

Cardiac Perfusion. Perfusion will be assessed using a routine Technetium(Tc-99 m) Sestamibi radionuclide scan at rest and after intravenousadenosine. The Emory Cardiac Toolbox will be used for imagequantification. Evaluation will use a 17-segment model. A core reviewlab will assess the perfusion studies with the interpreter blinded tothe study cohort. Improvements in perfusion will be expressed insemi-quantitative terms (yes/no). The percentage of patients observed tohave improvement in perfusion will be compared between dose cohorts.

MRI. Regional and global wall motion, infarct size, and left ventricular(“LV”) volumes will be measured using MRI. Subjects will receiveGadolinium contrast during scanning. MRI scan will use the breathholding technique. Steady state precession imaging to obtain global andregional LV function will be performed as will Gadolinium imaging. Leftventricular end systolic and diastolic volumes, LVEF, LV end diastolicdimension, wall thickness in systole and diastole of the infarctedregion, and infarct size will be reported using the AHA/AVV 17-segmentmodel with transmural extent of the infarct reported as <25%, 26%-50%,51%-75% and >76%. A core review laboratory will assess MM with theinterpreter blinded to the study cohort.

To be selected for this study, subjects must meet all of the followingclinical criteria (“inclusion criteria”):

-   -   Age: 18-75 years;    -   Acute ST segment elevation myocardial infarction meeting ACC/AHA        criteria, with symptoms of chest pain within 3 days of        admission. Criteria include (ST elevation >1 mm in limb leads or        2 mm in two or more precordial leads and increased levels of        troponin, creatine kinase MB (CPK MB) or both), New York Heart        Association (NYHA) heart failure class (to be recorded) of I, II        or III;    -   Eligible for percutaneous coronary intervention (PCI);    -   Eligible for MRI;    -   Eligible for Single Proton Emission Computed Tomography (SPECT)        imaging;    -   Subject must be able to provide informed written consent and        must be willing to participate in all required study follow-up        assessments;    -   Subjects must have a hemoglobin content (Hgb)>10 grams/dL, white        blood cell count (WBC)>3500 cells/mm³, a platelet count>100,000        cells/mm³ and an international normalized ratio (INR, a blood        coagulation test)<2.0 the day before the bone marrow collection;    -   Subjects must have a serum creatinine<2.5, total bilirubin<2.0        within 7 days of the bone marrow collection;    -   IRA and target lesion must be clearly identifiable when disease        is present in more than one vessel;    -   Successful reperfusion and intracoronary stent placement, with        Thrombolysis In Myocardial Infarction (TIMI) 2 or 3 flow and IRA        with <20% stenosis after revascularization;    -   Subjects must be deemed eligible to receive conscious sedation,        mini-bone marrow harvest, and second catheterization for        Chemotactic Hematopoietic Stem Cell Product infusion;    -   The type of stent used and time and date inserted must be        recorded;    -   Drug eluting stents should be limited to paclitaxel or sirolimus        types;    -   Included subjects must have an expected survival of at least one        year and must not have multiple vessel disease after        revascularization, or be expected to require intervention within        6 months of study entry.

Subjects who satisfy any one of the following criteria do not qualifyfor, and will be excluded from, the study (“exclusion criteria”):

-   -   Subjects who are not candidates for percutaneous intervention,        conscious sedation, MRI, SPECT imaging or mini-bone marrow        harvest;    -   History of sustained chest pain unrelieved by nitrates,        occurring 4 or more days before revascularization;    -   Subjects who fail to re-perfuse the infarct related coronary        artery or to have successful stent placement;    -   Subjects presenting with cardiogenic shock (systolic pressure<80        on vasopressors or intra aortic counterpulsation);    -   Subjects with a side branch of the target lesion>2 mm and with        ostial narrowing>50% diameter stenosis after revascularization;    -   Subjects unable to receive aspirin, clopidogrel or ticlopidine;    -   Subjects receiving warfarin must have an INR less than or equal        to 2; the term INR refers to INR International Normalized Ratio,        which is a system established by the World Health Organization        (WHO) and the International Committee on Thrombosis and        Hemostasis for reporting the results of blood coagulation        (clotting) tests;    -   Subjects with severe aortic stenosis;    -   Subjects with severe immunodeficiency states (e.g., AIDS);    -   Subjects with cirrhosis requiring active medical management;    -   Subjects with malignancy requiring active treatment (except        basal cell skin cancer);    -   Subjects with documented active alcohol and for other substance        abuse;    -   Females of child bearing potential unless a pregnancy test is        negative within 7 days of the mini-bone marrow harvest;    -   Subjects with ejection fractions greater than 50% on study entry        by SPECT (96 to 144 hours after stent placement);    -   Subjects with less than three months of planned anti-platelet        therapy post index procedure;    -   Subjects with multi vessel disease after revascularization        requiring subsequent planned intervention during the next 6        months;    -   Subjects with participation in an ongoing investigational trial;    -   Subjects with active bacterial infection requiring systemic        antibiotics.

Baseline assessments of cardiac function and cardiac perfusion will beobtained one day prior to the planned mini-bone marrow harvest andinfusion of the chemotactic hematopoietic stem cell product (see infra).A mini-bone marrow harvest (“MMH”) will be performed the day followingbaseline assessment of cardiac function and cardiac perfusion.

Example 2 Cardiac Catheterization

Sterile Preparation and Draping

The subject will be brought into the Cardiac Catheterization Laboratoryafter the investigator has obtained an informed consent. The subjectwill receive a sterile preparation and draping in the CardiacCatheterization Laboratory.

Cardiac Catheterization

Vascular access will be obtained by standard technique using right orleft groin. A sheath will be placed in the femoral artery or the rightor left brachial artery. Coronary arteriographic examination will beperformed by obtaining standard views of both right and left coronaryarteries. Multiple views will be obtained to identify the previouslystented infarct related artery. All subjects will receive standardmedications during the catheterization procedure in accordance withroutine practice.

Example 3 Acquisition Process for Acquiring Chemotactic HematopoieticStem Cell Product that is then Enriched for CD34+ Cells

While it is contemplated that any acquisition process appropriate foracquiring the chemotactic hematopoietic stem cell product comprisingpotent CD34+ cells is within the scope of the described invention, thefollowing example illustrates one such process referred to herein as amini-bone marrow harvest technique.

Preparation of Harvesting Syringes

Prior to the bone marrow harvest, forty 10 cc syringes loaded with about2-ml of a preservative free heparinized saline solution (about 100units/ml to about 125 units/ml, APP Cat. No. 42592B or equivalent) willbe prepared under sterile conditions. Heparin will be injected via asterile port into each of two 100-ml bags of sterile 0.9% normal salinesolution (“Normal Saline”, Hospira Cat. No. 7983-09 or equivalent)following removal of 10 cc to 12.5 cc of normal saline from each bag,resulting in a final heparin concentration of about 100 units/ml (U/ml)to about 125 units/ml (U/ml). 2-ml of the preservative free heparinsolution (about 100 U/ml to about 125 U/ml) will be loaded under sterileconditions into each of the forty 10 cc syringes, which then are cappedand placed into a sterile bag for transport to the harvesting site.

Subjects will be prepared for bone marrow harvest after written informedconsent is obtained as detailed in Example 1. Conscious sedation will beprovided using standard institutional procedures and guidelines. Bonemarrow harvest will be conducted under sterile conditions. The term“sterile conditions” as used herein includes proper scrubbing andgowning with a sterile mask and gloves worn by the harvesting attendingand assistant. The harvesting procedure can be performed outside of anoperating room as follows: after sterile prepping and draping, eachiliac crest should be anaesthetized with a 1% lidocaine solution using aminimum of 10-ml for each crest. The area of anesthesia should be acircular area no less than 10 cm in diameter. The harvesting needle isinserted until the iliac crest is punctured. The cap and stylet isremoved and 2-ml of marrow is harvested into the 10-mi harvestingsyringe containing 2-ml of the heparin solution. The syringe then isremoved and placed on the sterile field. After re-inserting the stylet,the harvesting needle is advanced slightly and then rotated 90°. Thestylet is then removed and an additional 2-ml of marrow is drawn intothe harvesting syringe retrieved from the sterile field. This procedureis repeated two more times until the harvesting syringe contains 8-ml ofmarrow for a total of 10-ml of heparinized marrow at a final heparinconcentration of about 20 U/ml to about 25 U/ml. Finally the fullharvesting syringe is handed to the harvesting assistant and shaken andinfused in the sterile collecting bag as described below. The harvestingphysician then takes the other harvesting needle that had been flushedpreviously with the heparin solution and repeats this process.

The full harvesting syringe is infused in the sterile collecting bag asfollows. The harvesting assistant is handed the full harvesting syringeand empties it in the 500-ml collecting bag though the sterile adaptorattached to the bag. Then the harvesting needle is flushed with theheparin solution in the flushing syringe and returned to the sterilefield.

The harvesting process is repeated on one iliac crest until about 19syringes have been collected and emptied in the collecting bag. The sameprocess is repeated on the other iliac crest until another about 19syringes have been filled. A total of thirty-eight 8 ml aspirations fromboth iliac crest (ideally 19 from each iliac crest) will result in302-ml of bone marrow harvested in a final volume of 380 ml at a heparinconcentration of about 20 U/ml to about 25 U/ml.

The collecting bag is sealed by tying off the connecting tube threetimes and then clamped distal to the ties. The bag is appropriatelylabeled “Human Bone Marrow Collection” and the results of the harvestingprocedure, including final volume collected and any procedure relatedcomplication, are recorded on the Mayo Clinical Risk Score (MCRS) casereport form. The completed label is affixed to the bone marrow bag. Thebag then is placed in a sterile carrying bag to be transported to theprocessing facility.

Example 4 Preparation of the Bone Marrow Product for Transportation

In one embodiment, the harvested bone marrow is transported to theprocessing facility as follows. When the clinical site is prepared toship the bone marrow preparation, 24-hour notice will be provided to theprocessing facility. The processing laboratory will make shippingarrangements at the earliest possible time for pickup for same daydelivery to the processing laboratory. Immediately after the bone marrowis collected, the bone marrow product will be placed in the suppliedshipping container. The shipping container contains two small blocks offrozen wet ice on the bottom and a sheet of bubble wrap on top of thewet ice. The bone marrow product is placed into a secondary bag and thesecondary bag is placed on top of the bubble wrap. A temperature tagmonitor (a sensor used to monitor the internal temperature) is affixedto the interior of the box. Another layer of bubble wrap then is placedon top of the product before the shipping container is sealed off.

Example 5 Selection of CD34+ Cells from the Harvested Bone MarrowProduct

CD34+ cells will be isolated from the harvested bone marrow product. Inone embodiment, CD34+ cells will be isolated using the anti-CD34monoclonal antibody (Mab), Dynabeads® M-450 Sheep anti-Mouse IgG, andPR34+(TM) Stem Cell Releasing Agent components of the Isolex 300iMagnetic Cell Selection System (Baxter Healthcare Corp. Cat. No. 4R9734)as described in U.S. Pat. Nos. 5,536,475, 5,035,994, 5,130,144,4,965,204, 5,968,753, 6,017,719, 6,251,295, 5,980,887, 6,676,937, U.S.Published Application No 2003/0232050, and the Isolex 300i PackageInsert, each of which is incorporated herein by reference. Thisoperating system has been adapted for isolation of CD34+ cells from bonemarrow according to the described invention.

Upon arrival at the processing laboratory, the harvested bone marrowproduct (in the collecting bag) is inspected immediately and the bagchecked for any leakage. The collection should be free flowing with noapparent clumps and should not be hemolyzed. The collection will not beused if the integrity of the bag has been breached in any way.

The bone marrow product should be processed within about 12 hours toabout 24 hours of inspection. A 300-ml or 400-ml transfer pack containeris obtained, and a plasma transfer set is attached to the sampling portof the container. The bone marrow product is transferred from thecollecting bag to the transfer pack container. The pooled bone marrowcollection product is mixed thoroughly by inverting the container twenty(20) times.

The pooled bone marrow collection product then is sampled for analysis.In one embodiment, a total volume of 2.0 ml of the product is removedand aliquoted as follows: 0.3 ml is used for a duplicate run of CompleteBlood Count (CBC) using a hematology analyzer; 0.2-ml is dispensed intoa 75×100-mm glass tube for the detection of Gram positive and Gramnegative bacteria by Gram Stain (Gram Stain Kit, VWR, Cat. NO.BB231401); as a sterility check, 0.6-ml is dispensed into a Tryptic SoyBroth (TSB) (VWR, Cat. No. 29446-184) bottle for aerobic bacteria growthassay, 0.6-ml is dispensed into a Fluid Thioglycollate Media (FTM) (VWRCat. #29446-138) bottle for anaerobic bacteria growth assay, and 0.3-mlis used in flow analysis for CD34+ cell enumeration and cell viability.

The collection is weighed on an electronic scale, and the appropriatetare weight of the collection bag recorded. The relationship of thevolume of the bone marrow product to the weight of the product can beexpressed asVolume (ml)=[Weight (gm) of product−Tare weight of bag (gm)]÷1.06(gm/ml)  (Formula 1)

The number of Total Nucleated Cells (MC) in the bone marrow product iscalculated using the white blood cell (WBC) count obtained from the CBCaccording to the following relationship:TNC=WBC/μl×1000×Product volume (ml)  (Formula 2)

The number of CD34+ cells in the bone marrow product is calculated fromthe following relationship:Total CD34+ cells in the bone marrow product=Number ofCD34+cell/μl×1,000×Product volume (ml)  (Formula 3)

The Red Blood Cell (RBC) volume of the bone marrow collection product iscalculated from the following relationship:RBC volume (ml)=Product volume (ml)×Hematocrit (%)/100  (Formula 4),

If the collection contains more than 20 ml of RBC, red blood celldepletion is required. RBCs are depleted by centrifugation.Centrifugation at 1000×g for 20 minutes at ambient temperature isperformed to separate the huffy coat from the RBCs. The term “buffycoat” refers to a thin grayish white fraction of a blood sample thatcontains most of the white blood cells (leukocytes). Immediately aftercentrifugation, a 60 ml syringe is connected to the bottom of thecentrifugation bag and the RBCs are removed. More than one syringe maybe needed to collect all the packed RBC. The RBC depleted bone marrowproduct then is washed to remove fat contents.

A 1-ml syringe is used to remove 0.3-ml of the RBC-depleted bone marrowcell product through the transfer set attached to the product bag and aCBC performed. The TNC of the RBC depleted bone marrow product isdetermined from the relationship:Total TNC of the RBC depleted product=WBC/μl of RBC depletedproduct×1000×180-ml  (Formula 5)

The TNC recovery of the RBC depleted product, which must be at least 80%of the original product count, is calculated from the relationship:TNC recovery=TNC of the RBC depleted product÷TNC of the unprocessedproduct×100%  (Formula 6)

The total RBC volume is calculated as described supra; the RBC volume inthe RBC depleted product should be less than <20-ml.

In one embodiment according to the described invention, the Isolex 300isystem is used to process the RBC-depleted product or the bone marrowproduct whose RBC volume is <20 ml according to the following processingsteps:

(i) The bone marrow is washed automatically to remove platelets;

(ii) CD34 positive (CD34+) cells are labeled specifically for selectionby incubation with the Isolex 300i CD34 monoclonal antibody (Mab);

(iii) Unbound reagent is removed by washing the cell suspension withbuffer solution;

(iv) Sensitized CD34+ cells (meaning CD34+ cells labeled with CD34 Mab)are captured by Dynabeads M-450 Sheep anti-Mouse Igo;

(v) A selection column is used to separate the magnetically-labeledDynabeads having captured CD34⁺ cells from unwanted cells, which arewashed through the selection column and collected in the NegativeFraction Bag; and

(vi) PR34+Stem Cell Releasing Agent releases CD34+ cells from thecolumn, and the CD34⁺ cells are collected in the End Product Bag. Thesystem performs several washing steps, disposing of most of the liquidinto the Buffer Waste Bag.

The Isolex(R) selected CD34+fraction is assayed as follows to determineWBC and CD34+ cell yields. The volume of the CD34 Positive Fraction isdetermined by mixing the cells in the End Product Bag; the bag is gentlymassaged by hand to ensure even cell distribution. A transfer set isinserted into the sampling port of the End Product Bag and a 60-mlsyringe attached. The cell suspension is withdrawn into the syringe(maximum 50-ml at a time) in order to measure the total volume.

A 3-ml or 5-ml syringe is used to remove a 2.0-ml sample from the EndProduct Bag through the transfer set for quality control testing. Thealiquoted volumes of the samples and the analyses performed on thosesamples are as previously described, i.e., CBC: 0.3-ml;

Gram stain: 0.3-ml; CD34+ cell enumeration and cell viability: 0.2-ml.

The total TNC of the CD34 Positive Fraction is calculated from therelationship:Total TNC of the Positive Fraction=WBC/μl of the PositiveFraction×1000×Volume of the Positive Fraction  (Formula 7)

The TNC recovery of the Positive Fraction, which must be less than 5% ofthe original product count, is calculated from the followingrelationship:TNC recovery=Total TNC of the Positive Fraction+Total TNC of theunprocessed product×100%  (Formula 8)

The total number of viable CD34+ cells in the Positive Fraction isdetermined from the following relationship:Total CD34+ cells in the Positive Fraction=Number of CD34+ cells/μl ofthe final product×1,000×Final product volume (ml)  (Formula 9)

The CD34+ cell recovery of the Positive Fraction s calculated from thefollowing relationship:CD34+ cell recovery=Total CD34+ cells of the Positive Fraction+TotalCD34+ cells of the unprocessed product×100%  (Formula 10).

Example 6 Preparation of Selected CD34+ Cells for Transfusion

Samples of the chemotactic hematopoietic stem cell product will beremoved to be assayed for WBC count, by flow cytometry (for CD34+ cellenumeration and viability), Gram stain, and sterility.

CD34+ cells are characterized by flow cytometric analysis featuringCD34bright and CD45dim fluorescence by double labeling with anti-CD34and anti-CD45 antibodies (Beckman Coulter, PN IM3630). CD34+ cells andCD45+cell viability is determined by excluding the dying cells whichtake up the intercalating DNA dye 7-aminoactinomycin D (7AAD). SeeBrocklebank A M, Sparrow R L. Cytometry. 2001; 46:254-261 (2001);Barnett D, et al. Br. J. Haematol. 106:1059-1062 (1999); Sutherland, etal., J Hematotherapy 5:213-226 (1996), and U.S. Pat. Nos. 4,520,110;4,859,582; 5,055,556; European Patent No. 76.695; Canadian Patent No.1,179,942 (PE, APC); U.S. Pat. No. 4,876,190 (PerCP); U.S. Pat. Nos.5,268,486; 5,486,616; 5,569,587; 5,569,766; 5,627,027 (Cy); U.S. Pat.Nos. 4,714,680; 4,965,204; 5,035,994 (CD34); U.S. Pat. No. 5,776,709(Lyse/no-wash method); U.S. Pat. Nos. 5,723,218 and 5,187,288 (TruCOUNTTubes), the contents of each of which is incorporated by referenceherein in its entirety.

Any flow cytometer or an equivalent device can be used for conductinganalysis of CD34+ cell enumeration and viability. In one embodiment, theprocessing laboratory employs a BD FACSCalibur™ flow cytometer and BDFACSComp™ software is used for instrument setup and monitoring. Atemplate and a panel of legend labels are preinstalled for acquisitionand analysis. Prior to use, the reagents, namely CD45FITC/CD34PE,Stem-Count Fluorospheres, Concentrated Ammonium Chloride Lysing.Solution, and 7AAD Viability Dye, are brought to ambient temperature.CD34+ cell controls are run as a positive control to affirm that theinstrument is set up for analyzing CD34+ cells, and the results arecompared with the manufacturer's pre-determined CD34 percent range.

The unprocessed bone marrow product and Isolex processed chemotactichematopoietic stem cell products may be analyzed by many differentprocedures. In one embodiment, or example, immediately upon receivingthe sample, if the WBC count of the sample is greater than 2×10⁷ cellsper ml, the sample is diluted with Sheath fluid to achieve a cell countof about 2×107 WBC per ml. 100 μl of the diluted product is aliquotedinto two 15×100 mm tubes. Using a micropipetter, 20 μl of CD45FITC/CD34PE and 7-AAD viability dye reagent are added into each tube and thesamples gently vortexed. The tubes are covered with aluminum foil andleft at ambient temperature for 15 to 20 minutes. RBCs are lysed byadding 1.5 ml of 1× Lysing Solution to each tube, vortexing gently. Thetubes are incubated for ten minutes at ambient temperature, protectedfrom light. The samples are stored at about 2° C.-about 8° C. (i.e., onan ice bath) protected from light until data acquisition is performed.Data acquisition must be performed within one hour of adding the lysingbuffer. Before data acquisition, Stem-Count Fluorospheres areresuspended by end-over-end rotation (10 times). 100 μl of Fluorospheresis added to each tube and gently vortexed taking care not to generateair bubbles. The absolute count of CD34+ cells in the product iscalculated from the relationship:Number of viable CD34+ cells per μl of product=LCD34×FAC  (Formula 11)

where LCD34 is the averaged number of events for Live CD34+/All CD 45+;“FAC” is Fluorospheres Assayed Concentration; and F is the averagednumber of Fluorosphere singlets counted.

The volume of CD34+Positive Fraction is calculated to obtain the numberof CD34+ cells required for the required dosing. The Required PositiveFraction Volume (ml) is defined as:The Requested CD34+ cell dosage (Total CD34+ cells per pi in thePositive Fraction×1,000).  (Formula 12)

An appropriate number of cells is dispensed into a 50 ml conical tubeand centrifuged at 500×g for 10 minutes. The supernatant is removedusing a 30 ml serological pipette and disposed of as waste whileexercising care not to disperse the cell pellets at the bottom of thetubes during this process. The infusion solution (20 ml) is added intothe CD34+ Cell Positive Fraction tube and the cells dispersed using a 10ml serological pipette by repeat pipetting. The resuspended cells arecentrifuged for 10 minutes at 500 g. A 30 ml serological pipette is used(without disturbing the cell pellet) to transfer thesupernatant/infusion solution into a 50 ml conical tube with a label“Positive Fraction Supernatant” affixed. The tube containing thesupernatant is vortexed to homogenize the solution. A 10 ml serologicalpipette is used to transfer 10 ml of the homogenized supernatant back tothe CD34+ Cell Positive Fraction tube. The remaining 10 ml of suspensionin the Supernatant tube will be used for sterility testing (5 ml eachinto a TSB (Trypticase Soy Broth) bottle and an FTM (FluidThioglycollate) bottle). The cells in the CD34+ Cell Positive Fractionare resuspended by slowly withdrawing and aspirating through a blunt endneedle affixed to a 10 ml syringe (Infusion Syringe) several times. Thecell suspension is withdrawn into the syringe, any air bubbles reaspirated off, and the blunt end needle removed. The infusion syringe isattached to the injection port of a 4-way stopcock.

The chemotactic hematopoietic stem cell product of the describedinvention will be released for infusion only if it meets the followingcriteria:

-   -   CD34⁺ cell purity of at least about 70%, 75%, 80%, 85%, 90% or        95%;    -   A negative Gram stain result for the selected positive fraction;    -   Endotoxin Levels: less than about 0.5 endotoxin units/ml;    -   Viable CD34⁺ cell yield of the “Chemotactic hematopoietic stem        cell product” meets the required dosing as per the treatment        cohort;    -   CD34⁺ cells are at least about 70%, 75%, 80%, 85%, 90% or 95%        viable by 7-AAD;    -   USP sterility result for “Positive Fraction Supernatant”:        negative (14 days later); and    -   Bone marrow CD34⁺ cell selection was initiated within about 12        hours to about 24 hours of completion of bone marrow harvest.

Sterility assessment on the stem cell product including gram stainingand endotoxin will be performed prior to product release for infusion.USP sterility (bacterial and fungal) culture will be performed and theresults will be reported to the principal investigator. In the event ofa positive USP sterility result, the subject and attending physician oncall will be notified immediately, provided with identification andsensitivity of the organism when available, and documentation ofappropriate anti-microbial treatment and treatment outcome will berecorded by the investigative site and the sponsor.

After meeting these release criteria, the chemotactic hematopoieticstern cell product will be released for infusion and packaged fortransportation to the catheterization facility. A sample also will besent for in vitro testing.

According to some embodiments, product will be released only if CD34+cell selection is initiated within 12 hours to about 24 hours ofcompletion of bone marrow harvest and only if it is to be infused withinabout 48 hours to about 72 hours of completion of bone marrow harvest.

According to some embodiments, the nonexpanded, isolated population ofautologous mononuclear cells containing CD34+ cells, which furthercontain potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotacticactivity is divided into aliquots, which are frozen at −86° C. andcryostored in the vapor phase of a liquid nitrogen freezer forsubsequent administration. Each of these aliquots can be used to preparea thawed chemotactic hematopoietic stem cell product as follows. Thefrozen nonexpanded, isolated population of autologous mononuclear cellsare thawed at a sufficient time before planned administration thesterile nonexpanded, isolated population of autologous mononuclear cellscomprising CD34+ cells, which further contain a subpopulation of potentCD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity willbe enriched for CD34+ cells, which further contain a subpopulation ofpotent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotacticactivity so as to yield the thawed chemotactic hematopoietic stem cellproduct. Samples of this thawed chemotactic hematopoietic stem cellproduct will be removed to be assayed for WBC count, by flow cytometry(for CD34+ cell enumeration and viability), Gram stain, and sterility.The thawed chemotactic hematopoietic stem cell product will be releasedfor infusion within about 48 hours to about 72 hours of thawing of thefrozen aliquot of the sterile nonexpanded, isolated population ofautologous mononuclear cells comprising CD34+ cells.

Example 7 Formulation of the Chemotactic Hematopoietic Stem Cell ProductComprising CD34+ Cells

The chemotactic hematopoietic stem cell product is formulated in 10-mlof saline (0.9% Sodium Chloride, Injection, USP, Hospira, Cat#7983-09)supplemented with 1% HSA (Human Albumin USP, Alpha, Cat. #521303)(“Infusion Solution”) and more than 20% autologous serum. In addition,there may be some trace amount of materials (quantities not determined)in the Chemotactic hematopoietic stem cell product that are used andleft over during the product processing. These materials include:Dulbecco's Phosphate Buffered Saline-Ca++, Mg++Free (D-PBS) (Baxter,Cat. #EDR9865), Sodium Citrate (Baxter/Fenwal, Cat. #4B7867), Hetastarch(Abbott Laboratories, Cat. #0074-7248-03), IVIg (Gammagard® ImmuneGlobulin Intravenous, Baxter, Cat. #060384) and the reagents in theIsolex® 300i Stem Cell Reagent Kit (Baxter, Cat. #4R9734) includinganti-CD34 monoclonal antibody, stem cell releasing agent and Sheepanti-mouse magnetic beads.

Example 8 Transporting Chemotactic Hematopoietic Stem Cell Product tothe Catheterization Facility

According to the original plan, the chemotactic hematopoietic stem cellproduct that met the release criteria was to be loaded into a sterile 10cc syringe in a Class 100 biological safety cabinet located within acontrolled aseptic environment, e.g., at minimum, a Class 100,000 cellprocessing facility; class 10,000 is preferable, but not required. Thechemotactic hematopoietic stem cell product was suspended in 10-ml PBSsupplemented with HSA and the container labeled in accordance withrelease criteria. The original plan called for four dosing cohortsconsisting of five subjects each in each cohort. The first was toreceive about 5×10⁶ CD34+ cells, the second about 10×10⁶ CD34+ cells,the third about 20×10⁶ CD34+ cells and the fourth about 30×10⁶ CD34+cells. Subjects in the higher dosing cohorts with inadequate CD34+ cellquantities to meet the assigned cohort dose were to be added to a priorcohort at the greatest possible CD34+ cell dose. The loaded infusionsyringe was attached to a four-way stopcock along with a flushingsyringe, capped and have safety guards applied to prevent leakage. Thedelivery apparatus was sealed in a double sterile bag and placed in asecure transportation box for transportation to the cardiaccatheterization facility. Following release of the chemotactichematopoietic stem cell product and cohort assignment, the chemotactichematopoietic stem cell product was shipped to the catheterization sitefor direct infarct-related artery infusion (“intravascularadministration”).

Example 9 Intra-Coronary Infusion of Chemotactic Hematopoietic Stem CellProduct

Upon notification from the cell processing facility that the chemotactichematopoietic stem cell product had been released for infusion (seesupra), the subject/patient was scheduled to arrive at thecatheterization facility at a time to coincide with the arrival of thechemotactic hematopoietic stem cell product.

Cardiac enzymes (brain natriuretic peptide (BNP), troponin and CPK MB),complete blood counts, a full chemistry panel (renal and liver functiontest) and an EKG were performed just prior to chemotactic hematopoieticstem cell product infusion. Clinical assessment of the stage of heartfailure according to the New York Heart Association's (NYHA) functionalclassification system was recorded.

Upon receipt of the chemotactic hematopoietic stem cell product andfinal quality assurance release (by facsimile) for infusion, the subject1 did undergo cardiac catheterization as detailed above. Coronaryarteriography was performed to assess for patency (meaning openness,freedom from blockage) of the infarct related artery and Thrombolysis inMyocardial Infarction (TIMI) angiographic flow. A balloon catheter overa wire was placed in the stented segment of the infarct related artery.Any appropriate balloon dilatation catheter having an internal diameterof at least about 0.36 mm compatible with the chemotactic hematopoieticstem cell product infusion can be used. After positioning, the balloonwire was removed. The chemotactic hematopoietic stem cell productdelivery apparatus was removed from the transportation case.

The delivery apparatus was in a sterile bag and had safety blocksattached to the infusion syringe (containing the chemotactichematopoietic stem cell product) and the flushing syringe. The apparatusconsisted of the infusion syringe (containing 10 ml of the chemotactichematopoietic stem cell product) and the flushing syringe (containing 6ml of flushing solution) wherein both were attached to a sterilefour-way stopcock. The entire delivery apparatus was shaken gently toresuspend the CD34+ cells in the infusion solution. The flushing syringewas used to eliminate all air bubbles in the apparatus (to prevent airemboli) and the delivery apparatus then attached to the balloondilatation catheter via the stopcock.

Delivery of the chemotactic hematopoietic stem cell product to thesubject by infusion proceeded as follows. First, with the stopcock openbetween the flushing syringe (6 ml solution) and the central lumen ofthe balloon catheter, 1 ml of flushing solution was infused (afterremoval of the guard) into the central lumen of the catheter over 15seconds. Second, the balloon was inflated at two atmospheres of pressurewithin the stent to avoid damage to the coronary artery endothelium andthen the stopcock valve adjusted to allow infusion of the chemotactichematopoietic stem cell product distal to the inflated balloon (afterremoval of the guard). With the balloon inflated, about 3 cc to about 4cc from the infusion syringe was infused by hand over a period of about30 seconds to about 45 seconds (to be timed and documented). The balloonremained inflated to allow adhesion of the CD34+ cells and to preventback flow for a total of about 2 minutes to about 3 minutes (includingthe time for infusion). In between infusions, the balloon remaindeflated for 3 minutes to allow restoration of blood flow (reperfusion).It was expected that 3 infusions will be required to empty the infusionsyringe. Third, upon completion of infusing the chemotactichematopoietic stem cell product and with the balloon deflated, the valveon the stopcock was adjusted to allow filling of the infusion syringefrom the flushing syringe. Finally, with the balloon inflated (about 2minutes to about 3 minutes), the 4 ml of flushing solution now in theinfusion syringe was infused over a period of about 30 seconds to about45 seconds to dislodge any residual CD34+ cells from the syringe andcatheter into the IRA circulation. The catheter then was removed.

An infusion-related ischemia (inadequate blood flow) assessment wasperformed during the first 24 hours after chemotactic hematopoietic stemcell product infusion. An EKG at about 12 hours and at about 24 hoursand analytical chemistry of cardiac enzymes (BNP, troponin and CPK MB)about every 8 hours for about 24 hours was obtained. Arrhythmiaassessment (24 hour Holter monitor) was performed immediatelypost-chemotactic hematopoietic stem cell product infusion.

All subjects were provided with digital thermometers and a log book torecord twice daily temperatures for 30 days post infusion of thechemotactic hematopoietic stem cell product. Subjects were instructed tonotify the investigator site immediately for temperatures recorded above100.5° F. Rapid follow-up with appropriate cultures and radiographicassessments was performed according to routine clinical standards.Documented bacterial infections, if any, were reported to the IRB andthe FDA.

Additional follow-up visits for safety assessments included visits at 1week and 2 weeks after product administration. Visit assessmentsincluded a comprehensive medical history and physical examination, EKG,complete blood counts, full chemistry panel (renal and liver functiontest), and measure of serum cardiac markers (BNP, troponin and CPK MB).Clinical assessment of NYHA functional class was recorded on week 1 and2. At 4 weeks post chemotactic hematopoietic stem cell product infusion,an EKG and cardiac enzymes (BNP, troponin and CPK MB) was obtained. A 24Holter monitor was used to assess for arrhythmias. Clinical assessmentof NYHA functional class was recorded. Treadmill exercise testing usinga symptom limiting Bruce protocol was performed as well.

At about 3 months and about 6 months post chemotactic hematopoietic stemcell product infusion at the first infusion time, a 24 hour Haltermonitor was performed. Clinical assessment of NYHA functional class wasrecorded. At about 6 months post chemotactic hematopoietic stem cellproduct infusion, a symptom limited treadmill exercise testing using theBruce protocol was recorded.

A safety assessment at about 12 months post chemotactic hematopoieticstem cell product infusion will include a comprehensive medical historyand physical examination, EKG, complete blood counts, full chemistrypanel (renal and liver function test), and measure of serum cardiacmarkers (BNP, troponin and CPK MB). A 24 hour Holter monitor will beperformed. Clinical assessment of NYHA functional class will berecorded.

Statistical Analysis

A paired design, where each subject serves as his or her own control,was used in some embodiments. Differences between before and aftertreatment, per subject, were analyzed for each of the four numericcardiac functions (i.e., myocardial contractility; end systolic volume,end diastolic volume; and perfusion). Linear regression analysis wasused to assess the significance of increased dosing levels. The nullhypothesis is that the slope of the regression line (dosing levelserving as the independent variable and the “after” minus the “before”difference serving as the dependant variable) is equal to zero. Thepower of rejecting a false null hypothesis is 0.68 at the 0.05 alphalevel of significance for a high correlation of 0.5 between dosing andimprovement in cardiac function. The 95% confidence interval about theslope of the regression line was used to assess the medical significanceof the increase in dosing level. If the slope of the regression line wasnot significantly different from zero but the intercept of theregression line is different from zero, then all treatment groups wascombined and a paired t-test will be performed to assess the overalltreatment effectiveness. The null hypothesis is that the mean of thedifferences is equal to zero. A Wilcoxon signed-ranks test also wasperformed as an additional test to determine the treatmenteffectiveness. This test is more powerful (rejecting a false nullhypothesis) than a t-test if the observations are not normallydistributed. The power of the t-test is 0.79 for rejecting a false nullhypothesis at the alpha level of 0.05 and the treatment having a mediumsize effect (an effect large enough to be discernable by the naked eye).The medical significance of the treatment effect size was determined bycomputing a 95% confidence interval about the mean of the differences(the true mean of the differences will lay in this interval in 95% oftested samples).

To assess improvement in perfusion, logistic regression was used withdosing level as the independent variable and perfusion change (1=yes,0=no) as the dependant variable. Odds ratios of the four dosing levelswas computed separately with 5.0×10⁶ cells serving as the index group.

A binomial test was used to assess the significance of CD34+ cell dosingon perfusion. It was expected that there would be no spontaneousimprovement in a perfusion defect if present on the baseline perfusionscan. Therefore, any clinically significant improvement in a perfusiondefect when assessed at 6 months and compared to baseline was considereda treatment effect.

A concurrent group (non-treated controls) meeting eligibility but notreceiving CD34+ cells was evaluated similar to the treated group andassessed for significant improvement in cardiac function/perfusion. Eachstudy site alternated accrual of treated and non-treated controls. Acoin flip was used to determine the initial (treated or non-treated)subject sequence at each site. Comparison of outcomes between treatedand non-treated groups was made. The core lab was blinded regardingtreatment or no-treatment.

An assessment was performed to determine if a correlation existedbetween clinical outcome and cell content (CD34+) and/or in vitro colonygrowth (CFU-GM, CFU-GEMM, BFU-E), CXCR-4 mobility, and CXCR-4 surfaceantigen expression.

As originally planned, a total of 20 subjects were to receive thechemotactic hematopoietic cell product of the described invention. Therewere to be four dose cohorts (about 5×10⁶, about 10×10⁶, about 20×10⁶,and about 30×10⁶ CD34+ cells). If the chemotactic hematopoietic stemcell product content in any subject was not sufficient for the assignedcohort, that subject was reassigned to a prior cohort at the greatestpossible dose. Subjects having fewer than 5×10⁶ CD34+ cells availablefor infusion were removed from the study, did not undergo repeatcatheterization and were not counted as part of the 20-subject studygroup. In addition, if the chemotactic hematopoietic cell product of thedescribed invention did not meet release criteria, the subject did notreceive the cell product and was not counted as a study candidate to bereplaced by the next subject. In any cohort dosing group, if a subjectexperienced an acute (meaning immediate to about 7 days post infusion)unexpected toxicity considered to (probably) be a result of the cellproduct infusion, dose escalation was halted and 3 additional subjectswere accrued to that dose level. If no other unexpected toxicity wasobserved, then dose escalation resumed, however the total of 20 subjectswas not exceeded. If another toxicity occurs at that dose level, thenall subsequent subjects were accrued to the next lower dose level.

The chemotactic hematopoietic stem cell product of the describedinvention was not administered to any subject in the higher dose cohortuntil all the subjects from the prior dose cohort had completed theirfollow-up assessments two weeks after product administration.

Example 10 Experimental Results of Preliminary Studies

A series of preliminary preclinical studies were performed in an attemptto accomplish the following goals:

(1) Optimize the manufacturing process for the Mini bone-Marrow Harvest(MMH);

(2) Evaluate the stability of the inbound MMH product and the outboundhematopoietic cell product.

(3) Evaluate the internal diameter allowance and safety of thecatheters;

(4) Evaluate the compatibility of the cell product with the cathetersintended to be used in the study; and

(5) Evaluate the suitability of using the supernatant of the finalhematopoietic cell product to represent the final hematopoietic cellproduct for stability testing.

Study 1: Optimizing The Manufacturing Process for the Mini Bone-MarrowHarvest (MMH)

The effect of key manufacturing variables on the yield of viable CD34cells from representative bone marrow products was evaluated. A total ofsix (6) volunteer donors over the age of 45 (based on a range of 45-57)and three under 30 years of age (based a range of 21-28) agreed todonate an average of 45 ml (based on a range of 31 ml-54 ml) bone marrowand provided written Informed Consent for the procedure. The marrowaspiration technique employed was identical to that to be performed forthe clinical scale MMH (see Example 3, supra). As shown in Table 2, thecell counts of nucleated cell (NC) and CD34+ cells of Mini bone-MarrowHarvest (“MMH”) derived cells collected from volunteer donors appearedto be age related.

TABLE 2 Effect of donor age on nucleated cell yield of the MMH. Donorage group Over 45 (45-57) Under 30 (23-28) Volume of Viability CD34cells Volume of Viability CD34 cells Donor MMH (ml) (%) (10⁵ per ml) MMH(ml) (%) (10⁵ per ml) 1 31.30 83.85 1.27 48.00 96.90 7.98 2 43.50 97.423.89 50.60 96.28 11.60  3 51.50 85.74 1.37 39.90 87.17 5.99 4 47.5080.95 1.76 — — — 5 53.70 98.21 5.58 — — — 6 44.90 96.36 4.48 — — — Avg.45.40 90.42 3.06 46.17 93.45 8.52

The average cell count of the bone marrow products from older donors(N=6) was 28.4×106 (based on a range of 15.8×10⁶-49.5×10⁶) nucleatedcells per ml [“NC/ml”], with an average viability, as determined by7-AAD dye exclusion and flow cytometry, of 90.42% (based on a range of80.95%-98.21%) and CD34+ content of 106×10⁵/ml (based on a range of1.27×10⁵/ml-5.58×10⁵/ml). In the younger subject group (N=3), theaverage cell count collected from marrow aspiration was 46.2×10⁶ NC/ml(based on a range of 39.9×10⁶ NC/ml-50.6×10⁶ NC/ml), with an average7-AAD viability of 93.5% (based on a range of 87.17%-96.90%) and totalCD34⁺content of 8.5×10⁵/ml (based on a range of 5.99×10⁵ CD34⁺cells/ml-11.60×10⁵ CD34⁺ cells/ml).

Red Cell Depletion and CD34 Selection

TABLE 3 CD34+ cell recovery after RBC depletion of MMH from older agegroup (4557) donors. Donor 1 2 3 4 5 Average Method of RBC HetastarchBuffy Buffy Buffy Buffy — depletion coat coat coat coat CD34⁺ cell %1.09 1.64 1.63 1.45 1.99 1.58 in MMH: Pre-RBC depletion CD34⁺ cell %1.33 1.55 1.51 1.61 1.84 1.57 in MMH: Post-RBC depletion CD34⁺ cell65.68 92.36 80.66 78.79 81.67 79.83 recovery post RBC depletion (%)

As shown in Table 3, following red cell depletion of the MMH-derivedbone marrow products collected from the older donors, an average of79.83% (based on a range of 65.68%-92.36%) of the CD34 cells from theinitial MMH was recovered. There was no significant difference betweenthe initial CD34 cell purity (1.58%, based on a range of 1.09%-1.99%)and that following red cell depletion (1.57%, based on a range of1.33%-1.84%). Assay methods to quantify chemotaxis are well known in theart, and a wide variety of techniques are used to evaluate chemotacticability of a variety of cell types. Furthermore, cell migration assaysare commercially available.

The assay used for the determination of in vitro migratory activity ofCD34+ cells mediated by CXCR-4, which is adapted from an assay describedin Jo et al (J. Clin. Invest. 105: 101-11 (2000)), relies ontransmembrane migration of CD34+ cells. Transmembrane migration of CD34+cells from the upper chamber to the lower chamber of a transwellpolystyrene plate (6.5 mm diameter, 5 um pore size, Costar) is inducedby SDF-1 placed in the lower chamber. The number of migrated viableCD34+ cells in the lower chamber then is determined by flow cytometryanalysis using CD34/CD45 antibodies and 7-AAD. Control spontaneousmigration of CD34+ cells is performed without SDF-1 in the lowerchamber.

The subpopulation of potent cells that (I) express CXCR-4 and (ii) haveCXCR-4 mediated chemotactic activity, expressed VEGFR-2 at very lowlevels (mean 0.84%, range 0 to 2.39%). Because the subpopulation ofpotent CD34+ cells co-expresses CXCR-4, {CXCR-4 co-expression; mean60.63%, median 52% range 31-98% of CD34+ cells, capable of migrating inan SDF-1 gradient} while less than 2.5% of the CD34+ cells co-expressesVEGFR-2, functionally, these cells are VEGFR-2-, i.e., VEGFR-2 is notwhat drives the cells into the peri-infarct zone.

TABLE 4 CD34+ cell recovery, purity, CXCR-4 migratory activity,viability and hematopoietic CFU growth immediately after Isolexprocessing of MMH from older age group (age 45-age 57) donors. Donor 1 23 4 5 Average Storage time 0 0 0 12 10.50 — (hours) at 4° C.-8° C. CD34⁺cell 32.36 29.09 15.31 43.60 40.20 32.11 recovery (%) CD34⁺ cell 76.7673.64 71.66 72.52 72.01 73.32 purity (%} CD34⁺ cell 98.49 93.80 97.3898.28 98.39 97.27 viability CD34⁺ cell 22.10 2.60 22.00 19.90 19.7017.26 CXCR-4 migratory activity (%) Hematopoietic 27.5 25.0 18.9 17.021.00 21.9 CFU/100 CF34⁺ cells cultured

As shown in Table 4, following CD34 selection using the Isolex system,which includes immunomagnetic Dynabeads(R) and anti-CD34 mAb, an averageof 32.11% (based on a range of 15.31%-43.60%) of the CD34 cells wasrecovered with an average purity of 73.32% (based on a range of71.66%-73.64%) and an average viability of 97.27% (based on a range of93.80%-98.49%). In addition, these CD34+ cells displayed an average of17.26% (based on a range of 2.60%-22.10%) CXCR-4 migratory abilityimmediately after selection and were capable of generating hematopoieticcolonies (21.89 colonies/1C CD34+ cells plated (based on a range of 17.0colonies/100 CD34+ cells plated-27.5 colonies/100 CD34+ cells plated) inMethoCult culture.

Study 2: Evaluation of the Stability of the Inbound Mini-bone MarrowHarvest and of the Outbound Chemotactic Hematopoietic Cell Product

A series of experiments, using healthy volunteers, was performed inorder to evaluate the stability of the inbound MMH and of the outboundchemotactic hematopoietic stem cell product of the described invention.Assessment of the functional viability of the inbound and outboundproducts was evaluated by cell viability (7-AAD), SDF-1/CXCR-4 mediatedCD34⁺ cell migration, and the ability to form hematopoietic colonies inmethylcellulose (CFU colony forming ability).

To evaluate the inbound product stability for shipping and logisticpurposes and for coordination with clinical schedules, MMH products werestored at 4° C. to 8° C. as indicated. To evaluate the outbound productstability for shipping and logistic purposes, the chemotactichematopoietic stem cell product comprising isolated CD34+ cells enrichedfollowing MMH was stored at 4° C. to 8° C. as indicated.

In preliminary studies, cells either were processed immediately ormaintained at 4-8° C. for 12 hours prior to processing to evaluate theimpact of shipping and logistic duration on the manufacture of asuitable cell product for infusion. Despite the duration of storageprior to processing (inbound product expiration), the results did notvary significantly (data not shown).

In another series of experiments, cells were stored at about 4° C. toabout 8° C. for 12 hours and about 24 hours prior to reassessment tosimulate products infused at about 36 hours and at about 48 hours,respectively, following MMH.

TABLE 5 CD34+ cell viability, growth and CXCR-4 migratory activity13-13.5 hours after Isolex processing of MMH. Donor 1 2 Average CD34+cell viability (%) 97.59 96.90 97.24 CD34+ cell CXCR-4 migratoryactivity (%) 7.70 7.50 7.60 Hematopoietic CFU/100 CD34+ cells cultured18.00 25.00 21.5

As shown in Table 5, the isolated CD34+ cells of the chemotactichematopoietic stem cell product had an average viability of 97.24%(based on a range of 96.90%-97.59%) and average CXCR-4-mediatedmigratory capacity of 7.60% (based on a range of 7.50%-7.70%). As shownin Table 6, after storage for an average of 26.3 hours (based on a rangeof 26.0 h-26.5 h), these cells had an average viability of 96.81% (basedon a range of 96.39%-97.22%) and an average CXCR-4-mediated migratorycapacity of 4.75% (based on a range of 4.50%-5.00%). Further, the cellsstill maintained their ability to generate hematopoietic colonies invitro.

TABLE 6 CD34+ cell viability, growth and CXCR-4 migratory activity26.0-26.5 hours after Isolex processing of MMH. Donor 1 2 Average CD34+cell viability (%) 97.22 96.39 96.81 CD34⁺CXCR-4⁺ cell CXCR-4 migratoryactivity 4.50 5.00 4.75 (%) Hematopoietic CFU/100 CD34+ cells cultured28.00 14.00 21.00

Thus, an average of 13.3 hours (based on a range of 13.0 h-13.5 h) afterCD34+ cell selection, representing 26.0-26.5 hr post-MMH, the CD34+ cellpopulation had an average viability of 97.24% (based on a range of96.90%-97.59%), with average CXCR-4 mediated migratory capacity of 7.60%(based on a range of 7.50%-7.70%). At an average of 26.3 hours (based ona range of 26.0 h-26.5 h) following MMH, the average viability of thecells was 96.81% (based on a range of 96.39%-97.2%) and maintained anaverage CXCR-4-mediated migratory capacity of 4.75% (based on a range of4.50%-5.00%).

Formulation of the composition of the described invention comprisingthis product occurred an average of 8 hours (8.63±1.80 N=4) hours afterMMH collection, and infusion occurred within 24 hours of MMH.

TABLE 7 CD34+ cell viability as a function of time after MMH: 12- hourin-dating and 48 hour outdating (all time points measured fromcompletion of MMH.) CD34⁺ cell viability (%) Time (h) after MMH Average(SD) A B C D (SD) 98.22 97.13 97.60 99.00 97.99 (0.29) 24 95.32 97.76 —— 96.54 (1.73) 33 91.92 96.32 95.90 80.00 91.04 (7.62)

In a subsequent experiment, four (4) MMH products (A-D) were collectedand stored at 4° C. for an average of 12.8 hours (based on a range of12.5 h-13.0 h) before the CD34+ cells were isolated by the Isolexprocedure. This group, representing the “12 hour in-date” group (meaningthat the product was formulated within the in-date time of about 12hours), was evaluated for functional viability out-date at “24 hours”(22.9 h±1.63, N=4), “33 hours” (33.38±1.11, N=2), and “48 hours”(48.33±0.82, N=4) post MMH harvest. The data, summarized in Tables 7-9,demonstrate that following MMH, the chemotactic hematopoietic stem cellproduct comprising enriched CD34+ cells maintains 1) high viability(>90.0% average viability, Table 7), 2) 76.85% (±21.66) of theirSDF-1/CXCR-4 mediated migratory ability (Table 8), and 3) their abilityto form hematopoietic colonies in vitro (Table 9), respectively.

Table 8 shows SDF-1/CXCR-4 mediated CD34+ cell migration (% migratingCD34+ cells) as a function of time after MMH: 12-hour in-dating and48-hour outdating (all time points measured from completion of MMH). Forthe purpose of determining the impact of time post-MMH on the migratoryability of the CD34+ cells, time point “X” was considered the referencepoint, as this was determined to represent the earliest time pointfollowing MMH at which cells reasonably could be expected to be returnedto the subject in a finished formulation. The remaining migratoryactivity at the following time points (Y=33 hours, Z=48 hours) wascalculated as percent migratory ability remaining following the 24 hour(X) time point.

TABLE 8 SDF-1/CXCR-4 mediated CD34+CXCR-4+ cell migration (% migratingCD34+ cells) as a function of time after MMH: 12-hour in-dating and48-hour outdating (all time points measured from completion of MMH).Migrating CD34⁺CXCR-4⁺ cells (%) Average Time (h) after MMH A B C D (SD)24 (X) 20.00 18.50 21.50 36.00 24    (8.09) % Remaining 100.00 100.00100.00  100.00  100.00  (0)   33 (Y) 21.80 10.50 — — 16.15  (7.99) *%Remaining 109.00 56.76 — — 82.88 (36.94) 48 (Z) 8.80 17.00 17.50 31.0018.58  (9.19) ^(@)% Remaining 44.00 91.89 81.40 86.00 75.85 (21.66) *=(Y ÷ X) × 100% ^(@)= (Z ÷ X) × 100%

Table 9 shows the number of colony forming units (CFU) per 100 viableCD34+ cells plated as a function of time after MMH: 12-hour in-datingand 48 hour-out-dating (all time points measured from completion ofMMH).

TABLE 9 CFU per 100 viable CD34+cells plated as a function of time afterMMH # of CFU per 100 viable CD34⁺ cells plated Time (h) Average afterMMH A B C D (SD) 24 13.00 30.00 37.00 39.00 29.75 (11.81) 33 12.00 34.00— — 23.00 (15.56) 48 15.00 30.00 20.00  8.00 28.25 (14.57)

In an attempt to extend both the in-date and out-date stabilityparameters for the chemotactic hematopoietic stem cell product of thedescribed invention comprising CD34+ cells from 12-hours (in-date) andfrom 48-hours (out-date) (12/48), respectively, to 24-hours (in-date)and 72-hours (outdate) (24/72), respectively, CD34 cells were purifiedabout 12 hours after MMH harvest (12 hour in-date) and about 24 hoursafter MMH harvest (24 hour in-date) and analyzed for functionalviability at about 48 hours and at about 72 hours total time from MMH totime of testing/anticipated infusion (48 hour out-date and 72 hourout-date, respectively). Specifically, the functional viabilitycharacteristics of two MMH/chemotactic hematopoietic stem cell productsof the described invention were evaluated at 48 hours and 72 hours. Theresulting data were further compared to the same indices derived at theprevious 12/48 time points (Tables 7-9).

Tables 10-12 show that at 33 hours (based on 32.5±0.71, N=2), 48 hours(based on one data point at 49 hours), and at 72 hours (based on 72.5h±0.71, N=2), the isolated CD34+ cells of the chemotactic hematopoieticstem cell product of the described invention maintained 1) over 90%viability (Table 10), 2) 102.19±32.69% of their SDF-1/VEGF/CXCR-4mediated migratory ability (Table 11), and 3) their ability to generatehematopoietic colonies in vitro (Table 12).

TABLE 10 CD34+ cell viability as a function of time after MMH: 24-hin-dating and 72-h outdating (all time points measured from completionof MMH) CD34⁺ cell viability (%) Time (h) after Average MMH A B (SD) 3398.00 99.00 98.50  (0.71) 48 — 97.00 97.00 (—) 72 91.00 97.00 94.00 (4.24)

TABLE 11 SDF-1/CXCR-4 mediated CD34+ cell migration (% population ofmigrated CD34+ cells as a function of time after MMH): 24-h in-datingand 72-h outdating (all time points measured from completion of MMH)Time (h) after Migrating CD34⁺ cells (%) MMH Average (SD) A B (range) 338.20 14.05 11.13 (2.93) % Remaining 100.00 100.00 100.00 (0.00) 48 —18.61 18.61 (—) % Remaining — 132.46 132.46 (—) 72 5.70 18.95 12.33(6.63) % Remaining 69.51 134.88 102.19 (32.69)

The % remaining ratios in Table 11 were determined as in Table 8 above.

TABLE 12 Number of CFU per 100 viable CD34+ cells plated as a functionof time after MMH: 24-h in-dating and 72-h outdating (all time pointsmeasured from completion of MMH) # of CFU per 100 viable Time (h) CD34⁺cells plated after MMH Average (SD) A B (range) 33 26.00 28.50 22.25 (1.25) 48 — 16.80 16.80 (—) 72 14.50 27.50 21.00 (6.5)

Further evaluation of the functional viability parameters of thechemotactic hematopoietic stem cell product comprising isolated CD34+cells of the described invention (“clinical product”) at 8 hours (8.6h±1.80, N=4), 12 hours (12.87 h±1.92, N=4), 32 hours (one time point at33.5 h), 48 hours (47.50 h±2.5, N=2), and 72 hours (71.5 h±0.50, N=2)after MMH shows that after 72 hours, the product retains its 1)viability (Table 13), 2) SDF-1/CXCR-4 mediated migratory ability (Table14) and 3) ability to form hematopoietic colonies in vitro (Table 15),equivalent to the 24-hour time point.

TABLE 13 Clinical Product Experience: CD34+ cell viability as a functionof time after MMH. CD34⁺ cell viability (%) Time (h) Average after MMH AB C D (SD) 8 98.30 99.08 90.00 96.45 95.96 (4.12) 12 98.89 96.96 99.0099.43 98.57 (1.10) 33 — 93.42 — — 93.42 48 — 93.15 91.58 — 92.37 (1.11)72 — 91.25 89.25 — 90.30 (1.48)

TABLE 14 Clinical Product Experience: SDF-1/CXCR-4 mediated CD34⁺ cellmigration (% migrating CD34⁺ cells as a function of time after MMH)Migrating CD34⁺ cells (%) Average Time (h) after MMH A B C D (SD) 12 (X)14.31 13.08 9.74 31.73 17.97 (11.34) % Remaining 100.0 100.0 100.0 100.0100.0  (0)   33 (Y) — 6.17 — —  6.17 *% Remaining — 47.17 — — 47.17 48(Y) — 4.88 8.21 —  6.55  (2.35) *% Remaining — 37.30 84.29 — 60.79(23.49) 72 (Y) — 3.7 6.6 —  5.15  (2.05) *% Remaining — 28.29 21.19 —24.74  (3.55) *= (Y ÷ X) × 100%

All remaining ratios were calculated as in Table 8 above.

TABLE 15 Clinical Product Experience: # of CFU per 100 viable CD34+cells plated as a function of time after MMH # of CFU per 100 viableCD34⁺ cells plated Time (h) Average after MMH A B C D (SD) 12. 98.1433.30 24.00 22.50 44.49 (36.09) 33 — 16.50 — — 16.5 48 — 19.56 20.50 —20.03 (0.66) 72 — 20.45 21.19 — 20.82 (1.10)

Based on these data, extension of the in-dating to 24 hours (from12-hours) and the out-dating to 72 hours (from 48 hours) for the CD34+cell clinical product of the described invention is justified.

FIG. 1 indicates the equivalence of the functional viability of thechemotactic hematopoietic cell product of the described invention at 72hours to the same indices evaluated at 48 hours.

Study 3: Catheter Safety.

The viability and potential efficacy of the chemotactic hematopoieticstem cell product of the described invention comprising potent CD34+cells depends on the cells maintaining their potency as they passthrough a catheter. The catheter used in the methods of the describedinvention has an internal diameter of at least 0.36 mm. Any type ofcatheter having an internal diameter of at least 0.36 mm may beeffective in delivering the pharmaceutical compositions of the describedinvention.

In one embodiment, the catheter is a balloon catheter. Balloon cathetersafety studies were conducted to determine whether high cellconcentrations and repeated perfusions adversely affect cell viability,cell recovery or catheter integrity, Non-mobilized peripheral bloodprogenitors were used in order to obtain an adequate number of cells toperform the analysis. Catheters were assessed for infusion of the cellproduct of the described invention comprising selected CD34+ cellsthrough the IRA. None of the 0.36 mm internal diameter catheters testedadversely affected CD34+ selected cell viability, growth in culture, ormobility in CXCR-4 assays.

TABLE 16 Viability of CD34⁺ cells before and after infusions through thecatheters. Viability (%) Catheter Condition 1 2 3 4 5 — Pre-infusion81.45 Raptor After 1st infusion 84.29 70.94 87.89 88.02 84.68 After 2ndinfusion 83.00 87.44 86.39 79.91 83.18 Sprinter After 1st infusion 93.3991.09 84.13 88.28 81.68 After 2nd infusion 91.89 91.08 84.88 77.65 77.73Voyager After 1st infusion 94.21 86.21 83.08 77.53 69.68 After 2ndinfusion 88.03 84.71 79.27 78.11 76.80 Maverick After 1st infusion 90.0089.76 90.79 85.49 81.31 After 2nd infusion 90.94 87.38 81.98 80.09 85.47

As shown in Table 16, in all catheters tested, average CD34+ cellviability was at or above 70% following passage through the catheters.

To demonstrate that infusion of the CD34+ cell product does not pose anysafety breach of the catheter used and that a significant percentage ofcell product does not adhere to the interior walls of the catheter,catheters were challenged with repeat infusions of a CD34+ cell producthaving a considerably higher cell concentration than that usedclinically. Four brands of catheters (Sprinter, Voyager, Maverick andRaptor) were evaluated using 5 catheters of each type. Non-mobilizedapheresis products were used in order to obtain an adequate number ofcells to perform the analysis. A cell concentration greater than threetimes that planned as treatment doses for the trial, i.e., 160×106nucleated cells containing CD34+ cells in 10 ml of infusion solution,was passed twice through each catheter. The average CD34+ cell recoverywas 100.59% (based on a range of 76.99% to 228.70%) following passagethrough the catheters.

All twenty catheters were tested for integrity using a methylene bluedye leak test after two perfusions with the nucleated cells. There wasno evidence of leakage and the contact points and catheter tips werenormal upon inspection.

As shown in Tables 17a and 17b, the effect on the cells of theirperfusion through a catheter appears to be independent of catheter modeland make among those catheters tested and was independent of the amountof time the cells were stored either prior to processing and/or afterCD34+ cell selection and prior to perfusion, resulting in a finalformulation containing an average recovery of 96.0% (range 80.8%-102.2%)of the CD34+ cells (Table 17b) and 86.36% of the CD45+cells perfusedthrough the catheter. Further, the average viability of the cells was96.5% (range 92.5%-98.6%, N=16); the cells maintained both CXCR-4migratory capacity (data not shown) and their ability to formhematopoietic colonies in methylcellulose (average 25.8 CFU/100 cellsseeded (range 21.0%-30.5%)

TABLE 17a CD45 cell recovery and viability after being infused throughthe catheters. 1 2 3 Catheter Condition Recovery R'd viab Recovery R'dviab Recovery R'd viab Raptor After 1^(st) infusion 69.68% −1.35% 78.67%2.08% 72.14% −4.55% After 2^(nd) infusion 97.91% −8.55% 81.84% −4.76%142.98% 3.28% Sprinter After 1^(st) infusion 76.74% −0.60% 68.56% 4.01%72.63% 5.29% After 2^(nd) infusion 78.82% 2.86% 85.40% 0.98% 90.29%−1.02% Voyager After 1^(st) infusion 87.38% 1.58% 83.93% −0.36% 103.58%0.93% After 2^(nd) infusion 82.70% 7.01% 69.34% 15.90% 69.54% 10.40%Maverick After 1^(st) infusion 73.97% 1.58% 87.01% 0.42% 78.31% 0.69%After 2^(nd) infusion 152.35% −5.06% 73.44% 2.78% 80.85% −3.92% 4 5Average Catheter Condition Recovery R'd viab Recovery R'd viab RecoveryR'd viab Raptor After 1^(st) infusion 80.54% 1.83% 73.21% −2.13% 74.85%−0.82%  (30.83%) (2.53%) After 2^(nd) infusion 107.82% −8.48% 94.08%0.08% 104.93%  −3.69%  (47.60%) (4.94%) Sprinter After 1^(st) infusion73.61% 6.06% 66.83% 8.31% 71.67% 4.61% (29.48%) (3.51%) After 2^(nd)infusion 82.22% 6.50% 91.61% 0.00% 85.67% 1.86% (35.30%) (2.78%) VoyagerAfter 1^(st) infusion 95.82% 4.52% 131.55% −4.39% 100.45  0.46% (44.39%)(2.91%) After 2^(nd) infusion 89.04% 0.27% 69.03% 7.50% 75.93% 8.22%(32.11%) (6.09%) Maverick After 1^(st) infusion 75.53% 2.61% 77.22%2.95% 78.41% 1.65% (32.33%) (1.21%) After 2^(nd) infusion 97.10% −2.97%91.11% −2.07% 98.97% −2.25%  (49.11%) (2.85%) Average of all catheters:86.36% 1.26% ^(a)Recovery of CD45+ cells = (# of CD45 cells afterinfusion + # of CD45 before infusion) × 100% ^(b)Reduction of CD45+ cellviability = [1 − (CD45+ cell viability % after infusion + CD45+ cellviability % before infusion)] × 100%

TABLE 17b CD34 cell recovery and viability after being infused throughthe catheters. Catheter 1 2 3 used Condition Recovery^(a) R'd viab^(b)Recovery R'd viab Recovery R'd viab Raptor After 1^(st) infusion 116.49%−3.48% 121.62% 12.91% 110.89% −7.91% After 2^(nd) infusion 91.66% 1.53%85.18% −23.26% 122.47% 1.71% Sprinter After 1^(st) infusion 89.19%−14.66% 83.34% −11.83% 102.72% −3.29% After 2^(nd) infusion 103.52%1.61% 99.82% 0.01% 82.11% −0.89% Voyager After 1^(st) infusion 81.02%−15.67% 96.08% −5.84% 90.16% −2.00% After 2^(nd) infusion 106.48% 6.56%81.66% 1.74% 95.04% 4.58% Maverick After 1^(st) infusion 76.99% −10.50%101.79% −10.21% 98.62% −11.46% After 2^(nd) infusion 228.70% −1.05%88.66% 2.65% 103.35% 9.70% Average Catheter 4 5 Recovery R'd viab usedCondition Recovery R'd viab Recovery R'd viab (SD) (SD) Raptor After1^(st) infusion 97.55% −8.06% 96.14% −3.97% 108.54%  −2.10%  (45.46%)(7.79%) After 2^(nd) infusion 111.33% 9.21% 98.96% 1.78% 101.92% −1.81%  (43.73%) (11.14%)  Sprinter After 1^(st) infusion 84.57% −8.39%88.65% −0.28% 89.69% −7.69%  (37.26%) (6.16%) After 2^(nd) infusion114.87% 12.05% 100.45% 4.84% 100.15% 3.52% (42.22%) (4.90%) VoyagerAfter 1^(st) infusion 82.73% 4.82% 89.32% 14.46% 87.86% −0.85%  (36.28%)(10.13%)  After 2^(nd) infusion 94.81% −0.75% 91.01% −10.23% 93.80%0.38% (39.12%) (5.86%) Maverick After 1^(st) infusion 112.58% −4.96%96.05% 0.18% 97.21% −7.39%  (41.34%) (5.34%) After 2^(nd) infusion89.35% 6.31% 117.63% −5.12% 125.54%  2.50% (73.48%) (5.33%) Average ofall catheters: 100.59%  −1.68%  ^(a)Recovery of CD34⁺ cells = (# of CD34cells after infusion + # of CD34 before infusion) × 100% ^(b)Reductionof CD34⁺ cell viability = [1 − (CD34⁺ cell viability % after infusion +CD34⁺ cell viability % before infusion)] × 100%

Collectively these experiments demonstrate that the serial passage of achemotactic hematopoietic stem cell product comprising CD34+ cellsthrough a cardiac catheter with an internal diameter of at least about0.36 mm does not adversely affect either catheter integrity or CD34+cell potency, i.e., CD34+ cell viability, CFU colony growth, or CD34+CXCR+mediated migratory capacity/mobility.

Study 4: Compatibility of the Cell Product with the Catheters

To further test the compatibility of the chemotactic hematopoietic stemcell product comprising CD34+ cells with each of the catheters that maybe used for delivery of the cell product in the study, cell productswere tested after multiple passages through each catheter type toevaluate the effects of extreme conditions of stress that would begreater than those expected during the treatment protocol.

At 48 hours post-MMH harvest, the chemotactic hematopoietic stem cellproduct comprising a range of about 5.73×106 CD34+ cells to about21.10×106 CD34+ cells (i.e., dosages reflective of the treatment cohort)obtained from individual donors was infused sequentially through threecatheters of the same brand, one type of catheter for each donor(Sprinter, Voyager or Maverick), and the cell product assessed for CD34+cell recovery, colony formation and viability.

TABLE 18 CD34+ cell recovery and sterility after sequential infusionsthrough the catheters. Catheter used Condition Parameter SprinterVoyager Maverick Pre-infusion CD34⁺ cell yield 9.72 × 10⁶ 2.11 × 10⁷5.73 × 10⁶ After 1^(st) CD34⁺ cell recovery 111%  103% 99% catheterAfter 2^(nd) CD34⁺ cell recovery 94% 104% 97% catheter After 3^(rd)CD34⁺ cell recovery 99%  99% 106%  catheter Sterility (aerobic andNegative Negative Negative anaerobic microbes)

As shown in Table 18, viable, colony forming cells were recovered in allexperiments for all three catheters tested (cell recovery 99%, 99% and106%).

As shown in Table 19, the average viability of the CD34+ cells afterpassing through the third catheter was 94.000% (based on a range of93.55%-94.40%) versus 96.01% (based on range of 94.18%-97.93%) of thepre-infusion cell product.

TABLE 19 CD34+ cell viability after sequential infusions through thecatheters. CD34⁺ cell viability Condition Sprinter Voyager MaverickAverage Pre-infusion 94.18% 95.91% 97.93% 96.01% After 1st catheter94.73% 96.31% 95.45% 95.50% After 2^(nd) Catheter 95.34% 95.72% 95.01%95.36% After 3rd catheter 93.55% 94.40% 94.04% 94.00%

As shown in Table 20, colony forming unit (CFU) growth derived from theCD34+ cells after passing through the third catheter was 95.27% (basedon a range of 43.47%-163.64%) of the infusion product (i.e., the infusedchemotactic hematopoietic stem cell product comprising CD34+ cells).

TABLE 20 CFU growth of CD34+ cells after sequential infusions throughthe catheters. CFU per 100 CD34⁺ cells cultured Condition SprinterVoyager Maverick Pre-infusion 30.5 11.5 11.0 After 1st catheter 22.014.0 22.0 After 2nd catheter 20.5 4.0 19.0 After 3rd catheter 24.0 5.018.0 Recovery from the pre- 78.69% 43.47% 163.64% infused product afterthe 3rd catheter Average recovery 95.27%

To determine the effect of catheter perfusion on CD34+ cell mobility andability to grow in culture, a series of experiments were performed whereMMH cells obtained from healthy donors were stored at 4° C. for 12 or 24hours before initiation of Isolex processing. Isolated CD34+ cellproduct that had been stored for about 12 hours pre-Isolex processingthen were stored at 4° C. until about 36 hours had elapsed from the endof processing, for a total of about 48 hours post MMH. At that time theywere assessed for SDF-1/CXCR-4 mobility and CFU growth pre and postperfusion through a 0.36 mm inner diameter (i.d.) cardiac ballooncatheter. Similarly, cells that were stored pre-Isolex processing for 24hours then were stored at 4° C. until 48 hours had elapsed from the endof Isolex processing, for a total of 72 hours, and then assessed.

TABLE 21 12 inbound/48 outbound and 48 hour inbound/72 hour outboundfrom MMH: SDF-1/CXCR-4 mobility (% population of migrated CD34+ cells)and CFU (per 100 viable CD34+ plated) pre catheter perfusion (“PRE”) andpost catheter perfusion (“POST”) Time (h) after SDF-1/CXCR-4 mobility(%)//# of CFU per MMH 100 viable CD34⁺ cells plated Inbound/outbound A BC D E 12/48 2.7//14  8.8//15 15.8//16 — — PRE 12/48 3.4//15 18.9//1317.6//8  — — POST 24/72 — — — 34//37 18.9//27.5 PRE 24/72 34//4323.5//24   POST

The results in Table 21 demonstrate that neither CD34+CXCR-4-mediatedcell mobility nor the cell's ability to grow in culture at any of thetime points tested was affected adversely by perfusion through acatheter having an internal diameter of at least 0.36 mm.

The Stabilizing Effect of Serum

The following data confirm the importance of the stabilizing effect ofserum to the migratory capability of the selected CD34+ cells.

As shown in Table 22, no CXCR-4 migratory activity was observed for allsamples tested including the pre-catheter infusion samples when thecomposition comprising a chemotactic hematopoietic stem cell product wasformulated without serum.

TABLE 22 Chemotaxis of CD34+ cells after sequential infusions throughthe catheters in the absence of serum. Migration (%) Condition SprinterVoyager Maverick Pre-infusion 0.0 0.0 0.1 After 1st catheter 0.0 0.0 0.0After 2nd catheter 0.0 0.0 0.1 After 3rd catheter 0.0 0.0 0.0

FIGS. 2 and 3 further illustrate that Isolex selected CD34+ cells retaintheir migratory capacity longer when formulated in the presence of humanserum. Following Isolex processing, the bone marrow derivedhematopoietic stem cell product comprising selected CD34+ cells wasformulated either in (1) phosphate buffered saline (Dulbecco's phosphatebuffered saline, Ca++, Mg++ Free (Baxter Cat. No. EDR9865) (“PBS”)containing 1% human serum albumin, 25 U/ml of heparin sodium and variousconcentrations (about 0%, about 10%, about 20%, or about 70%) ofautologous serum; or (2) normal saline (0.9%) containing 1% human serumalbumin, 25 U/ml of heparin sodium and (about 0% or about 10%)autologous serum. SDF-1/CXCR-4 mediated CD34+ cell migratory capacitywas evaluated at different times during final product storage (at 2°C.-8° C.) and after passing the cells through the catheter at the samerate and duration as anticipated by the clinical protocol. None of theseformulations affected CD34+ cell viability or the recovery of CD34+cells after they had been passed through the catheter.

Regardless of whether the chemotactic hematopoietic cell productscomprising selected CD34+ cells was (i) formulated either in PBS-serumor in saline-serum and (ii) either passed through the catheterimmediately or passed through the catheter after a prolonged stabilitytesting storage interval at about 4° C. to about 8° C., they maintainedan average of 96.6% viability (range 92.5%-98.6%) and an averageCXCR-4-mediated migratory capacity of 11.4% (range 2.4%-30.6%),representing a total time from harvest to mobility analysis of up to 48hours.

As shown in FIG. 2 panel (a), cells formulated in PBS alone at about 25hours retained about 10% of their CXCR-4 migratory capacity, whichdropped off to near 0 at about 48 hours. As shown in panel (b), cellsformulated in normal saline alone retained little, if any, of theirmigratory capacity. As shown in panels (c) and (d), cells formulatedwith PBS containing at least about 10% serum retained about 10-15% oftheir migratory capacity for up to about 55 hours (c), while cellsformulated with saline and at least about 10% serum retained about 20%of their migratory capacity for up to about 50 hours. As shown in panels(e) and (f), cells retained a higher migratory capacity for a longerduration in PBS supplemented with even higher concentrations of serum.

As shown in FIG. 3, the product of the described invention comprisingselected CD34+ cells when formulated in 10% serum, retained 14.25%, <1%,6%, and 5.8% of its CD34+CXCR4-mediated migratory capacity about 24,about 32, about 48 and about 56 hours after harvest, respectively. FIG.3 further shows that the product of the described invention comprisingselected CD34+ cells when formulated in 20% serum retained 18.25%,10.25%, 17% and 11% of its CD34+-CXCR-4-mediated migratory capacityabout 24, about 32, about 4S and about 56 hours after harvest,respectively. The term “stabilizing amount” as used herein thereforerefers to the amount of serum that, when included in the formulation ofthe product of the described invention comprising selected CD34+ cells,enables these cells to retain their CXCR-4 mediated chemotactic activityand hematopoietic colony forming ability.

As shown in Table 23, CD34+CXCR-4+ cells obtained from healthyvolunteers and from patients to which autologous serum was addedmaintained their motility out to 72 hours. CD34+ cells were isolatedfrom the bone marrow of healthy volunteers and of patients by themini-bone marrow harvest procedure as described in Example 3 underidentical conditions; and the chemotactic hematopoietic stem cellproduct was created as described in Examples 4 and 5. The products thenwere formulated with or without >20% autologous serum, and tested at 24,48 and 72 hours. As shown in column 2, CXCR-4 cell mobility ofCD34+CXCR-4+ cells obtained from healthy volunteers, when formulatedwithout serum, decreased 72% after 48 hours. As shown in column 3,CXCR-4 cell mobility of CD34+CXCR-4+ cells obtained from healthyvolunteers, when formulated with serum showed no change in meanCD34+CXCR-4+ cell motility, meaning that the serum stabilizesSDF-1/CXCR-4 motility. Column 4 shows that CD34+CXCR-4+ cells obtainedfrom patients showed less motility than did cells from healthyvolunteers, but that the motility of the CD34+CXCR-4+ cells wasmaintained out to 72 hours.

TABLE 23 Mean CD34+ Cell Mobility and % Change Over Time. Mean CD34+Cell Mobility % Volunteers With Hours Volunteers^(†) (N)^(††) Serum^(§)Patients With Serum 24 14.6 ^(|)(4) 18.1 (6) 12.8 (6) 48 3.2 (4) 19.7(8) 4.7 (3) 72 ND^(#) 22.1 (7) 4.6 (5) Mean % Change (range)** 48 ↓72(↓53-↓84) ↓0.6 (↓16-↑28) ↓57 (↓13-↓93) 72 ND ↑9.6 (↓30-↑85) ↓68(↓48-↓86) *Hours from bone marrow aspiration ^(†)CD34+ cells suspendedin PBS only ^(††)Number of individuals tested ^(§)CD34+ cells suspendedin PGS and autologous serum ^(|)% CD34+ migrating to lower chamber^(#)Not Done **Sum of % change of each experiment/number of experiments

Study 5: Final Product Sterility Testing

Due to the limited yield of CD34+ cells obtained from a 300-ml MMH,final cell product sterility is assessed using the supernatant removedfrom the final product formulation in order to preserve cell product forinfusion. Supernatant samples are loaded into the syringes in a manneridentical to that used to load the cell product into the syringes usedfor infusion (see supra).

To demonstrate that such a sample is representative of the final cellproduct formulation, we inoculated selected CD34+ cells in infusionsolution prior to centrifugation of the final product with C. sporogenes(13 CFU/ml), P. aeruginosa (2 CFU/ml), S. aureus (18 CFU/ml), A. niger(17 CFU/ml), C. albicans (3 CFU/ml) and B. subtilis (17 CFU/ml) (Seetable 24). After centrifugation, the sterility of both cell pellet andnon-cell supernatant fractions was assessed using USP aerobic andanaerobic testing.

TABLE 24 Bacteria and fungi used for the sterility study. Each sourcemicroorganism vial prepared by Microbiological Environments contained400 microbes per ml, but the numbers of CFU derived from each speciesare varied. Expected CFU/ml of Total # of inoculated sample Microbemicrobes/ml Total CFU/ml (21 ml) C. sporogenes 400 279 13 P. aeruginosa400 36 2 S. aureus 400 371 18 A. niger 400 356 17 C. albicans 400 62 3B. subtilis 400 349 17

As shown in Table 25, both the cell pellet fraction and suspensionfractions from all tested samples showed outgrowth of the inoculatedmicroorganisms, while un-inoculated controls showed no growth. Further,no apparent differential growth rate was observed between testing ofcell pellet fractions and the suspension fractions for allmicroorganisms tested. Samples taken before each step of the processingprocedure and following the final perfusion through the catheters alltested negative for microbial contamination.

TABLE 25 14-day sterility testing of nucleated cell (NC) samplesinoculated with specific species of microorganism (400 microbes in 21-m1NC sample). Sample with microbe Medium Sample Inoculated type fractionTest 1 Test 2 Test 3 C. sporogenes FTM^(a) Cell pellet Positive PositivePositive Suspension Positive Positive Positive S. aureus FTM Cell pelletPositive Positive Positive Suspension Positive Positive Positive P.aeruginosa FTM Cell pellet Positive Positive Positive SuspensionPositive Positive Positive A. niger TSB^(b) Cell pellet PositivePositive Positive Suspension Positive Positive Positive C. albicans TSBCell pellet Positive Positive Positive Suspension Positive PositivePositive B. subtilis TSB Cell pellet Positive Positive PositiveSuspension Positive Positive Positive Positive control: FTM CellPositive C. sporogenes Positive control: FTM suspension Positive S.aureus Positive control: FTM Positive P. aeruginosa Positive control:TSB Positive A. niger Positive control: TSB Positive C. albicansPositive control: TSB Positive B. subtilis Negative control: FTM CellNegative No microbes Negative control: TSB suspension Negative Nomicrobes ^(a)Fluid thioglycollate medium ^(b)Tryptic soy broth

Preclinical Study Summary

Collectively, these preclinical data indicate that the manufacturing andtesting procedures described are capable of generating adequate numbersof viable cells with adequate stability to withstand shipment andperfusion through the catheter in a manner that should pose noadditional safety concerns to the subject other than those associatedwith the routine use of fluid infusion through the balloon catheter.

Example 11 Preliminary Phase 1 Efficacy Data, with a Single InfusionDate

The following preliminary phase I efficacy data show that within about10×10⁶ isolated CD34+ cells, there are enough potent CD34+ cellsexpressing CXCR-4 and having CXCR-4-mediated chemotactic activity toeffect a paracrine effect, which affects immediate cell death and laterchanges consistent with ventricular remodeling.

In accordance with the disclosure in Example 1, a total of 31 subjectswere consented, eligible and enrolled in the study. The 31 patientsenrolled in the phase I study were randomly assigned to an autologousstem cell harvest treatment group or to a control group five days afteran ST elevation myocardial infarction (STEMI) characterized by aprolonged period of hypoperfusion (meaning blocked blood supply) Of the31 subjects enrolled, 16 were in the treatment group and 15 in thecontrol group. The first subject at each Center was randomized to eithertreatment or control, and each subsequent patient was enrolled intoalternating treatment or control groups. If the subject was assigned totreatment, he/she continued into the Treatment Phase as long as allinclusion/exclusion criteria continued to be met. Subjects assigned tothe control group progressed to the follow-up phase. There were nosignificant differences between groups in any of the baselinedemographic or clinical characteristics. Patients enrolled were from 34to 71 years of age, 87% male, 77% white, 61% in NYHA Class II or III and49% in NYHA Class 1, 74% experienced an infarcted left anteriordescending coronary artery, and 55% received a drug eluting stent.

CD34+ cells were isolated from the bone marrow by the mini-bone marrowharvest procedure as described in Example 3 within 5-8 days post stentreplacement. Harvested marrow then was shipped to the cGMP cellprocessing facility as described in Example 4 and isolated as describedin Example 5.

As originally planned, and as described in Example 8, there were to befour dosing cohorts (5 million, 10 million, 15 million and 20 millionCD34+ cells) in the study. However more than 15 million cells postCD34+selection could not be obtained reliably. Therefore enrollmentterminated at the end of cohort 3 with 15×106 being the highest celldose assessed.

Following cell product release and cohort assignment, the CD34+ cellproduct was shipped to the catheterization site for direct infarctrelated artery infusion. Treatment infusion occurred 6-9 days post stentreplacement (and within 48 hours of mini-bone marrow harvest). Subjectswere brought to the catheterization laboratory only after the CD34+ cellproduct had arrived at the facility and had received final release forinfusion.

The dosing cohorts consisted of 5 subjects in cohorts 1 and 2, 6subjects in cohort 3, and 15 control subjects. For cohort 1, thechemotactic hematopoietic stem cell product of the invention comprised5×10⁶ isolated CD34+hematopoietic stem cells containing a subpopulationof at least 0.5×10⁶ potent CD34+ cells expressing CXCR-4 and havingCXCR-4 mediated chemotactic activity [represented as “5 M”}. For cohort2, the chemotactic hematopoietic stem cell product of the inventioncomprised 10×10⁶ isolated CD34+ hematopoietic stem cells containing asubpopulation of at least 0.5×10⁶ potent CD34+ cells expressing CXCR-4and having CXCR-4 mediated chemotactic activity [represented as “10 M”].For cohort 3, the chemotactic hematopoietic stem cell product of theinvention comprised 15×10⁶ isolated CD34+hematopoietic stem cellscontaining a subpopulation of at least 0.5×10⁶ potent CD34+ cellsexpressing CXCR-4 and having CXCR-4 mediated chemotactic activity[represented as “15 M”}. Control subjects (i.e., those not receivingCD34+ cell infusion) were not expected to have significant improvementsin cardiac function (ejection fraction, end systolic and diastolicvolumes), or infarct region perfusion at 6 months follow up.

A sterile pharmaceutical composition of the described invention wasdelivered to each subject in cohorts 1, 2, and 3 parenterally byinfusion via the infarct-related artery through a catheter seven toeleven days following the STEMI. The sterile pharmaceutical compositioncomprised: (a) a therapeutically effective amount of a sterilechemotactic hematopoietic stem cell product, the chemotactichematopoietic stem cell product comprising an enriched population ofisolated CD34+ cells containing a subpopulation of potent cells havingchemotactic activity; which, when passed through the catheter remainedpotent, and (b) a stabilizing amount of serum.

Cardiac function follow-up was performed at 3 and 6 monthspost-infusion. Cardiac infarct region perfusion was assessed at 6 monthspost infusion. Both perfusion and functional follow-up testing wasassessed by a core lab facility blinded to the study treatment status ofeach subject. Comparison of these results to baseline indices wasperformed. Long term follow-up visits are conducted at 12 months andtelephone interview with subjects will be made annually at years 2through 5. For those subjects completing the 2-year follow-up telephonecall, no serious adverse events were reported, and thus, there have beenno long term safety events detected at this point.

The cardiac performance measures Resting Total Severity Score (RTSS),percent infarct (“% Infarct”), End Systolic Volume (ESV) and EjectionFraction (“EF”) were assessed at 3 months post treatment and at 6 monthspost treatment and compared with controls to assess efficacy of thecompositions compared to controls. Preliminary results are shown inTable 26. SPECT SCAN. As used herein, a single-photon emissioncomputerized tomography (SPECT) scan is a type of nuclear imaging test,which uses a radioactive substance and a special camera to create threedimensional images of the heart to show blood flows to the heart.Generally, the “Resting Total Severity Score (RTSS) is a score based onthe amount of dye not taken up in a SPECT SCAN. The data from RestingTotal Severity Score represents cardiac perfusion, i.e., blood flow atthe microvascular level, and muscle function. In brief, the technetiumdye used in a SPECT SCAN is taken up by the heart muscle. If the heartmuscle is healthy and takes up the dye, it appears white. If the heartmuscle is not healthy, dye uptake is diminished or does not occur atall, and the muscle appears gray to black.

Percent Infarct (MRI). The size of a heart attack matters fordetermining how well a patient will recover from the trauma. A patientwho has suffered damage to more than 30 percent of the left ventricle ofthe heart is twice as likely to die within a year from the injury as apatient who has suffered less damage, and bigger infarcts often requiremore aggressive therapy A computer method calculates the amount ofdamaged tissue by comparing MRI signal strength between damaged andundamaged tissue. Damaged heart tissue is denser than undamaged tissuebecause the muscle structure has collapsed, and MRI can distinguishbetween tissues of varying density. The term “percent (%) infarct” asused herein refers to the infarcted area compared to the rest of theheart. For purposes of this study, a % infarct greater than 20% isconsidered significant.

Preliminary results are shown in Tables 26 and 27. In order to assessstatistical significance, data for the control group and the 5 M groupwere pooled and data for the 10 M group and 15 M group were pooled (N=7for each pooled group). The preliminary results for these pooled groupsare shown in Table 27. Note that only the SPECT data reached statisticalsignificance; the other measures did not reach statistical significancebecause of the small numbers of patients involved.

TABLE 26 Phase I Efficacy Data 5M, 10M, 15M and Control QuantitativeMeasures of Left Ventricular Function Treated Treated Treated P-Value (5Million) (10 Million) (15 Million) All Treated All Treated CardiacFunction Test Control (N = 5) (N = 5) (N = 6) (N = 15) vs. Control MRI n= 10 n = 5 n = 4 n = 2 n = 11 n = 10 LVEF (%) Baseline 53 +/− 11 47 +/−13 47 +/− 11 50 +/− 7  48 +/− 10 6 Months 54 +/− 11 47 +/− 13 54 +/− 1150 +/− 6  50 +/− 11 Difference 1.1 +/− 7.8 −0.02 +/− 13    7 +/− 4 0.2+/− 0.8 2.5 +/− 9  0.706 EDV (mL) Baseline 154.7 +/− 55   153.3 +/− 30  176.6 +/− 51   175.7 +/− 12   165.8 +/− 36.1  6 Months 154.1 +/− 55  176.3 +/− 53   182.4 +/− 58   180.1 +/− 41   179.2 +/− 48   Difference−0.56 +/− 20    23.1 +/− 37  5.83 +/− 29  4.39 +/− 29  13.4 +/− 31 0.244 ESV (mL) Baseline 76 +/− 45 81 +/− 23 97 +/− 46 88 +/− 18 88 +/−30 6 months 74 +/− 44 95 +/− 46 87 +/− 46 91 +/− 32 91 +/− 40 Difference−1.84 +/− 17    14 +/− 25 −9.9 +/− 18  2.7 +/ 13   3.4 +/− 22  0.553Infarct Size¹ Baseline 17 +/− 8  18.8 +/− 8.6  33.2 +/− 14  12 +/− 1 22.8 +/− 13  (% of LV Mass) 6 months 10 +/− 9  16.2 +/− 10.9 22 +/− 1211 +/− 2  17.5 +/− 11  Difference −7 +/− 5  −2.6 +/− 5.9  −10.9 +/−3     −0.6 +/− 1    −5.2 +/− 6    0.570 SPECT n = 13 n = 5 n = 5 n = 4 n= 14 n = 13 RTSS Baseline 259 +/− 283 714 +/− 658 999 +/− 753 534 +/−440 779 +/− 620 (perfusion) 6 Months 273 +/− 395 722 +/− 521 636 +/− 532462 +/− 290 617 +/− 449 Difference  14 +/− 210  7.8 +/− 216 −363 +/−307  −122 +/− 260  −162 +/− 293  0.087 Combined Combined P-Value P-ValueControl Treated Control + 5 Control vs Control + 5 10 million + millionvs Cardiac Function Test 10 + 15 million million 15 million 10 + 15million MRI n = 6 n = 15 n = 6 LVEF (%) Baseline 51 +/− 11 48 +/− 9  6Months 52 +/− 12 53 +/− 9  Difference 0.336 1 +/− 9 4.5 +/− 5  0.352 EDV(mL) Baseline 154 +/− 47  176.4 +/− 39.9  6 Months 161.5 +/− 53.3  181.7+/− 48.7  Difference 0.617  7.3 +/− 28.1 5.35 +/− 25.9 0.884 ESV (mL)Baseline 78 +/− 38 94 +/− 37 6 months 81 +/− 44 88 +/− 38 Difference0.666 3.6 +/− 21  −5.7 +/− 17  0.341 Infarct Size¹ Baseline 17.3 +/−8.2  26 +/− 16 (% of LV Mass) 6 months  12 +/− 9.8 19 +/− 11 Difference0.794 −5.3 +/− 5.8  −7.5 +/− 5.7  0.450 SPECT n = 9 N = 18 n = 9 RTSSBaseline 385 +/− 450 814 +/− 636 (perfusion) 6 Months 398 +/− 465 559+/− 426 Difference 0.021  13 +/− 205 −256 +/− 298  0.011 ¹Infarct sizeexpressed in % of total mass (grams)

TABLE 27 Simple Changes in SPECT Rest Reperfusion Severity Score byTreatment Group - 6 Month Completers Change from P-value Baseline to 6Months 6 Month Baseline Post-Infusion Change Treatment Group 1 N 5 5Mean 714.200 7.8000 0.940 Std. Deviation 657.850 216.054 Minimum,(0.000, 1787.000) (−322.000, 222.000) Maximum Treatment Group 2-3 N 9 9Mean 814.333 −255.7778 0.033 Std. Deviation 635.641 297.644 Minimum,(97.000, 1868.000)  (−859.000, 263.000) Maximum Control Group N 13 13Mean 259.000 14.462 0.808 Std. Deviation 282.698 210.078 Minimum,(0.000, 858.000)  (−250.000, 528.000) Maximum Control + Treatment N 1818 Group 1 Mean 385.444 12.611 0.798 Std. Deviation 449.728 205.293Minimum, (0.000, 1787.000) (−322.000, 528.000) Maximum Note 1: p-valuesestimated from paired differences t-tests. 95% confidence interval fromt-distribution.

As for Resting Total Severity Score, Table 27 shows that for the 5M andcontrol pooled group, the change in Resting Total Severity Score after 6months was +12.6, indicating that the infarct area grew in thesepatients. The Resting Total Severity Score data further shows thatpatients in the 10M and 15M groups had bigger infarct areas at risk. The10 M and 15M group showed a drop of 31.4% in infarct size with a p of<0.01. Based on this data, infusion of at least 10×106 isolatedCD34+hematopoietic stem cells containing a subpopulation of at least0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediatedchemotactic activity results in a statistically significant improvementin infarct area perfusion.

The RTSS data for nontreated control subjects show neitherneoangiogenesis or prevention of cell death. When subjects were treatedwith a subtherapeutic dose of cells (i.e., 5×106 CD34+ cells containinga subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4and having CXCR-4 mediated chemotactic activity), RTSS data showedneither neoangiogenesis or prevention of cell death. Improvement in RTSSwas seen only in subjects treated with 10×106 or more CD34+ cellscontaining a subpopulation of at least 0.5×106 potent CD34+ cellsexpressing CXCR-4 and having CXCR-4 mediated chemotactic activity. Thisdose therefore is the minimal therapeutically-effective dose.

Example 12 Multiple Administrations of Chemotactic Hematopoietic StemCell Product to Subjects

The blood supply in the peri-infarct ischemic border zones is marginal,placing the cardiomyocytes of the border zone in jeopardy. Multipleinfusions of chemotactic hematopoietic stem cell product, by supportingcells in the border zone, can preserve/restore viability of theperi-infarct myocardium.

According to this aspect of the described invention, a first aliquot ofthe composition is administered at a first infusion date, a secondaliquot of the composition is administered at a second infusion date, athird aliquot of the composition is administered at a third infusiondate, and so on. The scheduling of infusion dates is determined for agiven patient by the treating practitioner according to his/her medicaljudgment.

According to one embodiment, the first infusion date is at least aboutone day, at least about two days, at least about three days, at leastabout four days, at least about five days, at least about six days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 15 days, atleast about 16 days, at least about 17 days, at least about 18 days, atleast about 19 days, at least about 20 days, at least about 21 days, atleast about 22 days, at least about 23 days, at least about 24 days, atleast about 25 days, at least about 26 days, at least about 27 days, atleast about 28 days, at least about 29 days, at least about 30 days ormore after occurrence of an AMI. According to another embodiment, thefirst infusion date is at least about 1 month, at least about 2 months,at least about 3 months, at least about 4 months, at least about 5months, at least about 6 months, at least about 7 months, at least about8 months, at least about 9 months, at least about 10 months, at leastabout 11 months, at least about 12 months, at least about 13 months, atleast about 14 months, at least about 15 months, at least about 16months, at least about 17 months, at least about 18 months, at leastabout 19 months, at least about 20 months, at least about 21 months, atleast about 22 months, at least about 23 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, at least about 72months, at least about 78 months, at least about 84 months, at leastabout 90 months, at least about 96 months, at least about 102 months, atleast about 108 months, at least about 114 months, at least about 120months, at least about 126 months, at least about 132 months, at leastabout 138 months, at least about 144 months, at least about 150 months,at least about 156 months, at least about 162 months, at least about 168months, at least about 174 months, at least about 180 months, at leastabout 186 months, at least about 192 months, at least about 198 months,at least about 204 months, at least about 210 months, at least about 216months, at least about 222 months, at least about 228 months, at leastabout 234 months, at least about 240 months or more after occurrence ofan AMI. According to some embodiments, the first infusion date is atleast 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years 17years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31years, 32 years, 33 years, 34 years, 35 years, 36 years 37 years, 38years, 39 years, 40 years or more after occurrence of an AMI.

According to another embodiment, the second infusion date is at leastabout one day, at least about two days, at least about three days, atleast about four days, at least about five days, at least about sixdays, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 16 days, at least about 17 days, at least about 18days, at least about 19 days, at least about 20 days, at least about 21days, at least about 22 days, at least about 23 days, at least about 24days, at least about 25 days, at least about 26 days, at least about 27days, at least about 28 days, at least about 29 days, at least about 30days or more after occurrence of an AMI. According to anotherembodiment, the second infusion date is at least about 1 month, at leastabout 2 months, at least about 3 months, at least about 4 months, atleast about 5 months, at least about 6 months, at least about 7 months,at least about 8 months, at least about 9 months, at least about 10months, at least about 11 months, at least about 12 months, at leastabout 13 months, at least about 14 months, at least about 15 months, atleast about 16 months, at least about 17 months, at least about 18months, at least about 19 months, at least about 20 months, at leastabout 21 months, at least about 22 months, at least about 23 months, atleast about 24 months, at least about 30 months, at least about 36months, at least about 42 months, at least about 48 months, at leastabout 54 months, at least about 60 months, at least about 66 months, atleast about 72 months, at least about 78 months, at least about 84months, at least about 90 months, at least about 96 months, at leastabout 102 months, at least about 108 months, at least about 114 months,at least about 120 months, at least about 126 months, at least about 132months, at least about 138 months, at least about 144 months, at leastabout 150 months, at least about 156 months, at least about 162 months,at least about 168 months, at least about 174 months, at least about 180months, at least about 186 months, at least about 192 months, at leastabout 198 months, at least about 204 months, at least about 210 months,at least about 216 months, at least about 222 months, at least about 228months, at least about 234 months, at least about 240 months or moreafter occurrence of an AMI. According to some embodiments, the secondinfusion date is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15years, 16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36years 37 years, 38 years, 39 years, 40 years or more after occurrence ofan AMI.

According to another embodiment, the third infusion date is at leastabout one day, at least about two days, at least about three days, atleast about four days, at least about five days, at least about sixdays, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 16 days, at least about 17 days, at least about 18days, at least about 19 days, at least about 20 days, at least about 21days, at least about 22 days, at least about 23 days, at least about 24days, at least about 25 days, at least about 26 days, at least about 27days, at least about 28 days, at least about 29 days, at least about 30days or more after occurrence of an AMI. According to anotherembodiment, the third infusion date is at least about 1 month, at leastabout 2 months, at least about 3 months, at least about 4 months, atleast about 5 months, at least about 6 months, at least about 7 months,at least about 8 months, at least about 9 months, at least about 10months, at least about 11 months, at least about 12 months, at leastabout 13 months, at least about 14 months, at least about 15 months, atleast about 16 months, at least about 17 months, at least about 18months, at least about 19 months, at least about 20 months, at leastabout 21 months, at least about 22 months, at least about 23 months, atleast about 24 months, at least about 30 months, at least about 36months, at least about 42 months, at least about 48 months, at leastabout 54 months, at least about 60 months, at least about 66 months, atleast about 72 months, at least about 78 months, at least about 84months, at least about 90 months, at least about 96 months, at leastabout 102 months, at least about 108 months, at least about 114 months,at least about 120 months, at least about 126 months, at least about 132months, at least about 138 months, at least about 144 months, at leastabout 150 months, at least about 156 months, at least about 162 months,at least about 168 months, at least about 174 months, at least about 180months, at least about 186 months, at least about 192 months, at leastabout 198 months, at least about 204 months, at least about 210 months,at least about 216 months, at least about 222 months, at least about 228months, at least about 234 months, at least about 240 months or moreafter occurrence of an AMI. According to some embodiments, the firstinfusion date is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15years, 16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36years 37 years, 38 years, 39 years, 40 years or more after occurrence ofan AML

Eligible subjects/patients presenting with symptoms and clinicalfindings suggestive of a myocardial infarction and eligible forinclusion in the study will be selected as described in Example 1 andcatheterized as described in Example 2. In some embodiments, thenonexpanded, isolated population of autologous mononuclear cellscomprising potent CD34+ cells will be acquired from the subject/patientas described in Example 3 and, in some embodiments, the harvested bonemarrow will be transported to the processing facility as described inExample 4. CD34+ cells will be selected from the harvested bone marrowproduct as described in Example 5.

The Isolex 300i system will be used to process the RBC-depleted productor the bone marrow product whose RBC volume is <20 ml according to thefollowing processing steps:

-   -   (i) The bone marrow is washed automatically to remove platelets;    -   (ii) CD34 positive (CD34+) cells are labeled specifically for        selection by incubation with the Isolex 300i CD34 monoclonal        antibody (Mab);    -   (iii) Unbound reagent is removed by washing the cell suspension        with buffer solution;    -   (iv) Sensitized CD34+ cells (meaning CD34+ cells labeled with        CD34 Mab) are captured by Dynabeads M-450 Sheep anti-Mouse IgG;    -   (v) A selection column is used to separate the        magnetically-labeled Dynabeads having captured CD34+ cells from        unwanted cells, which are washed through the selection column        and collected in the Negative Fraction Bag; and    -   (vi) PR34+ Stem Cell Releasing Agent releases CD34+ cells from        the column, and the CD34+ cells are collected in the End Product        Bag. The system performs several washing steps, disposing of        most of the liquid into the Buffer Waste Bag.

The Isolex(R) selected CD34+ fraction then will be assayed to determineWBC and CD34+ cell yields as described in Example 6. A first aliquot ofthe chemotactic hematopoietic stem cell product containing at least10×10⁶ CD34+ cells will be formulated a described in Example 7,transported to the catheterization facility as described in Example 8,and infused into the patient as described in Example 9 at the firstinfusion date. A plurality of aliquots of the nonexpanded, isolatedpopulation of autologous mononuclear cells containing CD34+ cells, whichfurther contain a subpopulation of CD34+/CXCR-4+ cells that haveCXCR-4-mediated chemotactic activity will be frozen at −86° C. andcryostored in the vapor phase of a liquid nitrogen freezer forsubsequent administration. (see “Cryopreservation Study” below).

CryoPreservation Study.

This study was conducted to evaluate the ability of the Isolex-basedportion of the chemotactic hematopoietic stem cell product manufacturingprocess to effectively enrich for CD34+ cells of the cryopreserved MMH.The protocol has been designed to evaluate the yield, viability,functionality and stability of the CD34+ cells derived from theenrichment of cryopreserved MMH. The study has been designed to evaluateand describe the effect on the chemotactic hematopoietic stem cellproduct of cryopreservation of the RBC reduced MMH prior to theIsolex-based CD34 selection.

The following experimental conditions have been applied:

(1) Two (2) MMH for each of three (3) replicates in order to provide foradequate cell yield to meet with requirements of the experimentaldesign; with a twenty four (24) hour interval between MMH andcommencement of RBC depletion procedure.

(2) Study control: Freshly prepared chemotactic hematopoietic stem cellproduct, with full product characterization after perfusion of thechemotactic hematopoietic stem cell product through a catheter at 48 and72 hours after MMH.

(3) Experimental: the chemotactic hematopoietic stem cell productderived from cryopreserved MMH, with full product characterization afterperfusion of the chemotactic hematopoietic stem cell product derivedfrom cryopreserved MMH through a catheter at 48 and 72 hours after MMH,minus the time the cryopreserved MMH remains in storage (defined as >24hours)

Study Design

In order to yield sufficient CD34+ cells to perform the intendedexperiment, two (2) donors will be required. More than or equal to 80 mlMMH and ≧30 ml of peripheral blood will be collected from each donor.

In bound storage: Samples will be stored at 2 to 8° C. for twenty four(24) hours before commencing the RBC reduction procedure.

Following RBC reduction, the MMH from both donors will be pooled andthen divided into two equal fractions. One fraction will be used as afresh (unfrozen) product control and the other fraction will be used forthe cryopreservation test.

For the cryopreservation test, RBC reduced MMH will be frozen in a −86°C. freezer and then cryostored in the vapor phase (≦−150° C.) of aliquid nitrogen freezer (LNF) using the cryoprotectant containing theliquid source Hetastarch (6% Hetastarch in 0.9% Sodium Chloridemanufactured by Hospira).

Both control (unfrozen) and cryopreserved (after thaw) samples will beIsolex processed essentially as described in Example 5 above. Samples intwo 10 ml syringes will be prepared from the selected CD34+ cells. Fullproduct characterization will be performed at the following time points:(i) After perfusion of the product through a catheter at 48 hours afterMMH; and (ii) after perfusion of the product through a catheter at 72hours after MMH. For the cryopreserved samples, the term “72 hours ofcollection”, for example, means the time from collection to the time oftesting, excluding the time elapsed from freezing and cryostorage of theRBC depleted bone marrow.

Key determinants for the CD34+ cell quality of the hematopoietic stemcell product include: (i0 CD34+ cell enumeration and 7-AAD viability;(ii) SDF-1/CXCR-4 mediated CD34+ cell migratory activity; (iii)expression of CXCR-4 cell surface antigen on CD34+ cells; and (iv)growth of hematopoietic progenitor cell colonies (CFU). This experimentwill be repeated three times.

Summary Of Results

The study was conducted in accordance with the methods described above.All deviations from methodology and materials used are detailed in therelated result sections presented below.

Table 28 summarizes the relevant information on the donors of the bonemarrow used in this study.

TABLE 28 Age and gender of the bone marrow donors for thecryopreservation study. Exp 1 Exp 2 Exp 3 Donor 1 2 3 4 5 6 Age 26 26 2262 32 24 Gender F F F F F F

Table 29 summarizes the sample volume, RBC content and the yield,viability and purity of the cells in the pre-processed MMH following24-h storage in a 2-8° C. refrigerator.

TABLE 29 Post 24 hours storage at 2-8° C. - Volume, cell yield andquality of MMH. Exp 1 Exp 2 Exp 3 Donor 1 Donor 2 Donor 3 Donor 4 Donor5 Donor 6 Volume (ml) 117 64 106 105 103 113 WBC per μl^(#) 1.39E+041.26E+04 1.39E+04 1.44E+04 1.94E+04 2.45E+04 TNC^(#) 1.62E+09 8.03E+081.47E+09 1.51E+09 1.99E+09 2.76E+09 HCT^(#) 33.85% 33.40% 29.10% 27.85%31.60% 32.60% RBC vol. (ml)^(#) 39.44 21.38 30.85 29.24 32.55 36.84CD45+ cell viability* 91.13% 91.72% 90.58% 93.17% 94.11% 95.8% ViableCD34+ cell 149.18 148.38 140.89 114.45 150.80 203.76 per μl* CD34+ cellviability* 94.14% 98.90% 98.35% 97.24% 98.89% 98.78% CD34+ cell purity*1.44% 1.32% 1.23% 0.97% 1.21% 0.88% CXCR-4 expressing 77.68% 77.03%71.88% 64.57% 75.75% 68.36% CD34+ cells^(~) Total # of CD34+ 1.74E+079.50E+06 1.49E+07 1.20E+07 1.55E+07 2.30E+07 cells* ^(#)Determined byhematology analyzer *Determined by flow cytometric analysis ofCD45-FITC/CD34-PE antibodies and 7-AAD staining of the sample^(~)Determined by flow cytometric analysis of CD34-FITC and CXCR-4-PEantibodies staining of the sample

In each of the experiments, the MMH from each pair of donors were pooledfollowing RBC reduction.

Table 30 presents the RBC content, viability and cell recovery of pooledMMH after RBC reduction:

TABLE 30 Post RBC reduction - RBC content and cell quality Exp 1 Exp 2Exp 3 Donors 1 & 2 Donors 3 & 4 Donors 5 & 6 RBC volume 15.35 ml 13.80ml 20.85 ml TNC recovery# 76.95% 85.93% 89.37% CD45+ cell viability84.97% 93.35% 95.60% CD34 recovery# 72.89% 84.00% 88.36% CD34+ cellviability 93.99% 97.92% 98.95% CXCR-4 expressing 71.33% 64.89% 74.64%CD34+ cells #As compared to the pre-processed samples

Following RBC reduction, each of the pooled MMH samples was divided intotwo equal fractions. One was used as a fresh (unfrozen) control and theother one was used for the cryopreservation test.

For cryopreservation, MMH mixed with an equal volume of chilledcryoprotectant was loaded evenly into two 250 ml Cryocyte containers,frozen in a mechanical freezer (−86° C.) and then stored cryopreservedin the vapor phase of a LNF according to the Protocol. Table 31 presentsdata obtained from post-thawed and washed MMH:

TABLE 31 Post thawed & washed MMH - Cell quality and recovery of cellsCryopreserved Sample Thawed and washed Exp 1 Exp 2 Exp 3 Storageduration prior 10 days 8 days 8 days to thaw Wash media PBS working 2%Dextran~ 8.3% Dextran@ sol'n* RBC volume 0.39 ml 1.11 ml 0.38 ml TNCrecovery# 36.11% 50.73% 28.61% CD45+ cell viability 61.85% 32.18% 43.97%CD34+ cell recovery# 52.43% 46.29% 15.72% CD34+ cell viability 94.36%86.11% 81.76% CD34+ cell purity  2.40%  1.29%  1.88% CXCR-4 expressing51.42% 50.74% 37.85% CD34+ cells Key: #As compared to the RBC reducedMMH before cryopreservation. *PBS Working Solution contained 1% HSA and0.41% sodium citrate (w/v) in PBS (Ca⁺⁺ and Mg⁺⁺ free). Washing of cellswith this solution was performed according to that instructed in theProtocol. ~This wash solution contained 2% Dextran 40, 1% HSA and 0.4%Na citrate in PBS (Ca⁺⁺ and Mg⁺⁺ free). The thawed sample was expandedwith 200 ml of this solution and was then washed twice each with 200 mlof this solution. Centrifugation was set for 600 g, 10 minutes at 20° C.The washed cells were resuspended with 150 ml PBS Working Solution forIsolex process. @This solution contained 8.3% Dextran 40 and 4.2% HSA insaline. The washing procedure was essentially as described for the 2%Dextran 40 wash solution.

Table 32 summarizes the CD34+ cell quality and recovery of thechemotactic hematopoietic stem cell product prepared from the unfrozenand cryopreserved MMH following Isolex processing.

TABLE 32 Post Isolex - Cell quality and recovery of cells Exp 1 Exp 2Exp 3 MMH source Unfrozen Frozen Unfrozen Frozen Unfrozen Frozen CD34+cell 47.28% 37.88% 35.94% 49.29% 44.05% 82.25% recovery# CD34+ cellviability# 99.37% 96.89% 98.97% 95.05% 98.26% 95.38% CD34+ cell purity87.51% 83.95% 86.47% 81.91% 81.71% 50.87% Total # of viable 4.63E+061.95E+06 4.07E+06 2.58E+06 7.50E+06 2.20E+06 CD34+ cells #As compared tothe RBC reduced sample for unfrozen samples and post thawed and washedsamples for frozen samples.

Following Isolex processing of each RBC reduced MMH pooled pair, twochemotactic hematopoietic stem cell product (“AMR-001”) samples withequal number of CD34+ cells, each in a 10 ml syringe, were prepared.Both AMR-001 samples were stored at 2-8° C. for stability testing. At 48and 72 hours from MMH (For cryopreserved MMH samples, the time forcryostorage was not included), a prepared AMR-001 was perfused through aballoon dilatation catheter performed in a manner as for a clinicalAMR-001. A full CD34+ cell characterization was performed on theperfused AMR-001 samples and the results are presented in Tables 33, 34,35, and 36. Table 37 shows the balloon dilatation catheter used.

TABLE 33 Post infusion through catheter - CD34+ cell purity, viabilityand recovery Catheter perfused AMR-001 MMH source Unfrozen Frozen Timepost MMH Experiment 48 h 72 h 48 h 72 h 1 CD34+ cell recovery# 101.73% 92.32% 91.71% 69.35% CD34+ cell viability 99.08% 98.13% 94.98% 91.80%CD34+ cell purity 85.92% 84.93% 82.94% 74.24% Total # of CD34+ cells2.36E+06 2.14E+06 8.92E+05 6.74E+05 2 CD34+ cell recovery# 95.65% 89.20%77.10% 74.01% CD34+ cell viability 98.29% 97.29% 89.47% 82.82% CD34+cell purity 81.49% 82.42% 75.30% 70.50% Total # of CD34+ cells 1.95E+061.81E+06 9.96E+05 9.56E+05 3 CD34+ cell recovery# 104.17%  101.99% 77.35% 79.12% CD34+ cell viability 98.46% 97.51% 86.86% 85.59% CD34+cell purity 83.18% 82.80% 47.81% 43.71% Total # of CD34+ cells 3.91E+063.83E+06 8.52E+05 8.71E+05 #As compared with the prepared AMR-001 beforeperfusion

TABLE 34 Post infusion through catheter - CXCR-4 expressing CD34+ cells(% of total CD34+ cells). MMH source of AMR-001 samples Catheter Exp 1Exp 2 Exp 3 perfusion Unfrozen Frozen Unfrozen Frozen Unfrozen Frozen 48h post MMH 66.52% 53.31% 57.64% 41.35% 60.14% 54.16% 72 h post MMH73.87% 53.89% 56.73% 44.07% 64.60% 50.67%

TABLE 35 Post infusion through catheter - Migratory CD34+ cells (% oftotal viable CD34+ cells). MMH source of AMR-001 samples Catheter Exp 1Exp 2 Exp 3 perfusion Unfrozen Frozen Unfrozen Frozen Unfrozen Frozen 48h post MMH 18.81 ± 1.83%* 5.87 ± 1.98% 19.67 ± 10.43% 15.67 ± 2.24%24.89 ± 1.93% 26.66 ± 1.53% 72 h post MMH (1.07%)# (1.51%) (1.06%)(2.19%) (1.44%) (1.56%) *SDF-1 induced migration. % of migratory CD34+cell of total viable CD34+ cells with standard deviation of threereplicates. #Natural migration (no SDF-1 added)

TABLE 36 Post infusion through catheter - Number of CFU per 100 viableCD34+ cells cultured. MMH source of AMR-001 samples Exp 1 Exp 2 Exp 3perfusion Unfrozen Frozen Unfrozen Frozen Unfrozen Frozen 48 h post MMH24 15.5 31.5 14 38 15.5 72 h post MMH 20.5 0.05 62.5 12 30.5 7

TABLE 37 Balloon dilatation catheters used MMH source Time of of theperfusion AMR-001 (Hours Balloon Exp sample of MMH) Manufacturelength/dia. Catalog # Lot # Comment 1 Unfrozen 48 h Sprinter 12/3.5 mmSPR3512W 258795 Outdated 72 h Sprinter 12/4.0 mm SPR4012W 254243Outdated Frozen 48 h Sprinter 15/3.0 mm SPR3015W 412090 Outdated 72 hVoyager 15/3.0 mm 1009443-15 8111462 — 2 Unfrozen 48 h Sprinter 15/3.5mm SPR3515W 443152 Outdated 72 h Sprinter 15/3.5 mm SPR3515W 443152Outdated Frozen 48 h Voyager 15/3.0 mm 1009443-15 8111462 — 72 h Voyager15/3.0 mm 1009443-15 8092561 — 3 Unfrozen 48 h Voyager 15/3.0 mm1009443-15 8111462 Reused* 72 h Sprinter 15/3.0 mm SPR3015W 476734Outdated Frozen 48 h Sprinter 15/3.0 mm SPR3015W 476734 Outdated 72 hSprinter 15/3.0 mm SPR3015W 476734 Outdated *Prior to be used for the2^(nd) time, the catheter and the central lumen were 1^(st) washed andflushed with 70% isopropyl alcohol and then with sterile PBS. Thecentral lumen was then injected with air in order to remove the residualliquid inside. The washing procedure was performed inside a bio-safetycabinet.

Discussion

The aim of this study was to evaluate the quality of AMR-001manufactured from cryopreserved MMH.

Post Isolex CD34+ cell recovery of the AMR-001 manufactured fromunfrozen MMH (Control samples) was on average 34.6±4.35% (range 30.3% to39%) which is within the acceptance range for manufacture of AMR-001 forclinical use. It should be noted that the data presented above areestimated without taking account for the cells removed for thein-process tests, therefore the actual CD34+ cell recovery will beslightly higher than that presented.

Post catheter CD34+ cell recovery was 100.52±4.39% (95.65% to 104.17%)at 48 hours post MMH and 94.50±6.67% (89.20% to 101.99%) at 72 hourspost MMH. There was no substantial reduction in viability (Table 33),CXCR-4 expression (Table 34), migratory activity (Table 35) and CFUgrowth (Table 36) of CD34+ cells at 72 hours post MMH as compared tothose monitored at 48 hours post MMH.

For the cryopreservation test, RBC reduced MMH samples werecryopreserved according to PCT protocol for cryopreservation of bonemarrow for transplantation where MMH samples mixed with equal volume ofcryoprotectant with final concentration of 5% DMSO, 2.5% HSA and 2.1%Hetastarch (from liquid source 6% Hetastarch, Hospira) were frozen at−86° C. and then cryostored in the vapor phase of a LNF.

Post cryopreservation and thaw, the stability, viability, mobility andgrowth in culture of Isolex selected CD34+ cells is maintained. Thus thefrozen-thawed cells meet the criteria for clinical use.

In some embodiments, a chemotactic hematopoietic stem cell productprepared from frozen and thawed aliquots of a sterile nonexpanded,isolated population of autologous mononuclear cells comprising CD34+cells, which further contain a subpopulation of potent CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity will be used forinfusion. Samples of thischemotactic hematopoietic stem cell productwill be removed to be assayed for WBC count, by flow cytometry (forCD34+ cell enumeration and viability), Gram stain, and sterility. Thechemotactic hematopoietic stem cell product will be released forinfusion within about 48 hours to about 72 hours of thawing of thesterile nonexpanded, isolated population of autologous mononuclear cellsonly if it meets the following criteria:

-   -   CD34+ cell purity of at least about 70%, 75%, 80%, 85%, 90% or        95%;    -   A negative Gram stain result for the selected positive fraction;    -   Endotoxin Levels: less than about 0.5 endotoxin units/ml;    -   Viable CD34+ cell yield of the “Chemotactic hematopoietic stem        cell product” meets the required dosing as per the treatment        cohort;    -   CD34+ cells are at least about 70%, 75%, 80%, 85%, 90% or 95%        viable by 7-AAD;    -   USP sterility result for “Positive Fraction Supernatant”:        negative (14 days later).

Sterility assessment on the stem cell product including gram stainingand endotoxin will be performed prior to product release for infusion.USP sterility (bacterial and fungal) culture will be performed and theresults will be reported to the principal investigator. In the event ofa positive USP sterility result, the subject and attending physician oncall will be notified immediately, provided with identification andsensitivity of the organism when available, and documentation ofappropriate anti-microbial treatment and treatment outcome will berecorded by the investigative site and the sponsor.

The chemotactic hematopoietic stem cell product prepared from the frozenand thawed autologous mononuclear cells will be formulated a describedin Example 7, transported to the catheterization facility as describedin Example 8, and infused into the patient as described in Example 9.

It is proposed that administration of a potent dose of CD34+/CXCR-4+cells that have CXCR-4-mediated chemotactic activity, early or lateafter occurrence of an acute myocardial infarction according to thedescribed invention canresult in a reduction in persistent/chronic andprogressive adverse cardiac events, including, but not limited to,premature death, recurrent myocardial infarction, the development ofcongestive heart failure, significant arrhythmias, and acute coronarysyndrome, and the worsening of congestive heart failure, significantarrhythmias, and acute coronary syndrome.

Example 13 Co-administration of the Chemotactic Hematopoietic Stem CellProduct and Neuregulin 1

Neuregulin 1 (NRG1) is an agonist for receptor tyrosine kinases of theepidermal growth factor receptor family, consisting of ErbB1, 2, 3, and4. (Fuller, S J, et al., J. Mol. Cell Cariol. 44: 831-54 (2008). Bindingof NRG1 to Erb4 increases its kinase activity and leads toheterodimerization with erbB2 or homodimerization with ErbB4 andstimulation of intracellular signal transduction pathways. Id. NFRG1receptor subunits ErbB2 and ErbB4 also are expressed in differentiatedcardiomyocytes. Id. Recently it has been shown in mice that NRG1 inducesproliferation of differentiated mononucleated cardiomyocytes in vivo byinducing differentiated cardiomyocytes to leave proliferativequiescence. Bersell, et al (Bersell, K. et al., Cell 138: 257-70 (2009).Undifferentiated stem and progenitor cells did not contribute to thisproliferation. (Id). Using a mouse model in which the left anteriordescending coronary artery (LAD) of two month old mice was ligatedpermanently and NRG1 administered daily one week later for 12 weeks, itwas shown that administration of NRG1 for 12 weeks resulted in asustained improvement in myocardial function, determined by ejectionfraction, a reduced infarct scar size, and attenuation of cardiomyocytehypertrophy. (Id).

Following acute myocardial infarction, in addition to necrotic celldeath as a consequence of ischemia, ongoing apoptotic cell death andcardiomyocyte hibernation collectively lead to a decrement in cardiacfunction that can worsen over time and ultimately causing major adversecardiac events. Once lost, cardiomyocytes are unable to significantlyregenerate to restore cardiac function. Carbon 14 dating ofcardiomyocytes show the regenerative capacity of cardiac muscle to beless than 1% annually (Bergman O, Science. 2009; 324:98-101). Thedescribed invention demonstrates the prevention of cardiomyocyte lossafter AMI through enhancement of perfusion and prevention of apoptosis.Further restoration of cardiac function requires significantlyincreasing the regenerative capacity of cardiomyocytes. Regeneratingcardiomyocytes will require adequate perfusion or will suffer theconsequences of ischemia including hibernation and apoptosis.

It is proposed that the combination of the described invention withsignificant augmentation of the natural regenerative capacity ofcardiomyocytes would be synergistic in restoring cardiac function afterAMI and preventing major adverse cardiac events. Co-administrationtherefore of the chemotactic hematopoietic stem cell product of thedescribed invention with neuregulin 1 is proposed as a therapeuticcapable of restoring cardiac function after AMI through increasingperfusion, which prevents apoptotoic cardiomyocyte cell death andrescues cardiomyocytes from hibernation, and by providing theinfrastructure needed for generation of new cardiomyocytes to replacelost cardiomyocytes.

Recombinant human neuregulin 1 will be obtained from commercial sources.(Cell Sciences, Novus Biologicals, R & D Systems, Raybiotech, Inc.,Shenandoah Biotechnology, Spring Bioscience).

Increased doses of neuregulin 1 will be admixed with the chemotactichematopoietic stem cell product of the described invention and tested invitro after passage through a catheter for product viability, sterility,purity and potency, meaning viability, migratory capacity andCFU-growth, after storage for up to 72 hours. If potency, purity andviability are maintained, a preclinical experiment is proposed in whichpurified, sterile human derived CD34+ cells containing a subpopulationof potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediatedchemotactic activity will be infused via the tail vein in Nod SCID miceafter coronary artery ligation and relief (induced AMI model). Theeffect of this treatment on cardiac perfusion, cardiac muscle function,histopathology, apoptosis, and scarring will be assessed post infusionand compared to controls (i.e., Nod SCID mice not receiving cells).Prior studies have demonstrated an improvement in perfusion, humanneoangiogenesis, prevention of apoptosis, and preserved cardiac functionin treated versus control animals. Next, increasing doses of neuregulin1 will be added to the purified, sterile human derived CD34+ cellscontaining a subpopulation of potent CD34+ cells expressing CXCR-4 andhaving CXCR-4-mediated chemotactic activity of the described inventionand the results will be compared to control animals and to animalstreated with the purified, sterile human derived CD34+ cells containinga subpopulation of potent CD34+ cells expressing CXCR-4 and havingCXCR-4-mediated chemotactic activity of the described invention alone.

If pre-clinical models show a potential synergistic beneficial effectwith the purified, sterile human derived CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingCXCR-4-mediated chemotactic activity of the described invention combinedwith neuregulin 1, a dose escalation safety and efficacy trial insustaining and in AMI patients is proposed. For this study, patientswill receive the purified, sterile human derived CD34+ cells containinga subpopulation of potent CD34+ cells expressing CXCR-4 and havingCXCR-4-mediated chemotactic activity of the invention with or withoutneuregulin 1. Neuregulin 1 will be administered in increasing doses todetermine (i) the mean therapeutic dose (MTD) and (ii) whether perfusionand cardiac function are enhanced by the combination of neuregulin 1 andthe purified, sterile human derived CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingCXCR-4-mediated chemotactic activity of the described invention comparedto the purified, sterile human derived CD34+ cells containing asubpopulation of potent CD34+ cells expressing CXCR-4 and havingCXCR-4-mediated chemotactic activity of the described invention alone.

While the described invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the describedinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of treating a progressive myocardialinjury that is an adverse consequence of a vascular insufficiencyaffecting coronary circulation of a subject, comprising (a) first,acquiring from the subject, under sterile conditions, a population ofmononuclear cells comprising CD34+cells; (b) second, sterilely enrichingthe population of mononuclear cells for CD34+cells, so that at least 70%of the cells comprising CD34+cells; (c) third, at least 24 hours afterstep (a), passing a first portion of the enriched population produced instep (b) through a catheter and confirming that (i) at least some of thecells passed through said catheter have CXCR-4-mediated chemotacticactivity and move in response to SDF-1 in vitro; (ii) at least some ofthe cells passed through said catheter can form hematopoietic coloniesin vitro; and (iii) at least 70% of the cells passed through saidcatheter are viable; (d) fourth, formulating for parenteral deliverythrough a catheter a sterile pharmaceutical composition comprising: (i)a second portion of the enriched population produced in step (b), whichcontains at least 0.5×10⁶ potent CD34+/CXCR-4+cells that haveCXCR-4mediated chemotactic activity and that move in response to SDF-1;and (ii) a stabilizing amount of serum of at least 10% relative to thevolume of the sterile pharmaceutical composition, wherein thestabilizing amount is effective to retain the CXCR-4-mediatedchemotactic activity and hematopoietic-colony-forming activity of theCD34+/CXCR-4+cells in the second portion of (d)(i); (e) fifth,administering the sterile pharmaceutical composition to the subjectparenterally on a first infusion date; and (f) sixth, repeating steps(b)-(d), in order, and optionally step (a), at a plurality of infusiondates to treat ongoing vascular insufficiency affecting coronarycirculation, thereby improving myocardial perfusion and preservingexisting myocardiocytes and their function during the subject'slifetime.
 2. The method according to claim 1, wherein the acquiring fromthe subject in (a) is early after occurrence of an acute myocardialinfarction.
 3. The method according to claim 2, wherein the acquiringfrom the subject in (a) is after peak inflammatory cytokine cascadeproduction in an infarcted area.
 4. The method according to claim 1,wherein the acquiring from the subject in a (a) is late after an acutemyocardial infarction.
 5. The method according to claim 4, whereinacquiring from the subject in (a) is at least 15 days after an acutemyocardial infarction.
 6. The method according to claim 1, wherein thevascular insufficiency is an ischemia.
 7. The method according to claim6, wherein the ischemia is a myocardial ischemia.
 8. The methodaccording to claim 1, wherein the vascular insufficiency of the coronaryvasculature develops after an acute myocardial infarction.
 9. The methodaccording to claim 8, wherein the first infusion date comprises aspecific time interval defined by a first time and a second time, andwherein the first time is after peak inflammatory cytokine cascadeproduction in an infarcted area and the second time is before myocardialscar formation in infarcted area.
 10. The method according to claim 9,wherein the first time of the first infusion date is at least about 5days post-infarction.
 11. The method according to claim 8, wherein thetherapeutic amount is effective to treat cardiomyocyte cell death in theperi-infarct border zone, hypoperfusion in the peri-infarct border zone,myocardial hibernation in the peri-infarct border zone, or a combinationthereof, relative to controls.
 12. The method according to claim 8,wherein the therapeutic amount is effective to decrease infarct area,decrease infarct mass, or a combination thereof, relative to controls.13. The method according to claim 8, wherein the progressive myocardialinjury is a progressive decline in heart muscle function following anacute myocardial infarction.
 14. The method according to claim 8,wherein the at least one adverse consequence of the vascularinsufficiency of the coronary vasculature that develops after an acutemyocardial infarction is a component of disease progression to heartfailure, wherein the disease progression to heart failure includesrecurrent myocardial infarction, development of significant arrhythmias,development of acute coronary syndrome, or a combination thereof. 15.The method according to claim 1, wherein the progressive myocardialinjury is heart failure.
 16. The method according to claim 1, whereinthe catheter has an internal diameter of at least about 0.36 mm.
 17. Themethod according to claim 1, wherein the administering is through thecatheter into myocardium, through the catheter intravascularly, or acombination thereof.
 18. The method according to claim 1, wherein thepharmaceutical composition further includes at least one active agentthat is compatible both with the CD34+cells, which further contain asubpopulation of potent CD34/CXCR-4cells that have CXCR-4-mediatedchemotactic activity and that move in response to SDP-1 and with thestabilizing amount of serum of the composition.
 19. The method accordingto claim 18, wherein the active agent is selected from the groupconsisting of, a cytokine, a diuretic, an anti-arrhythmic agent, atyrosine kinase receptor agonist, an anti-anginal agent, a vasoactiveagent, an anticoagulant agent, a fibrinolytic agent, and ahypercholesterolemic agent.
 20. The method according to claim 19,wherein the tyrosine kinase receptor agonist is human neuregulin
 1. 21.The method according to claim 9, wherein a second infusion date is atleast 30 days after occurrence of the acute myocardial infarction. 22.The method according to claim 1, wherein the mononuclear cells in (a)are cellular components of a bone marrow aspirate.
 23. The methodaccording to claim 1, wherein the mononuclear cells are cellularcomponents of peripheral blood.
 24. The method according to claim 9,wherein a second infusion date is at least 30 days after occurrence ofan acute myocardial infarction.
 25. The method according to claim 1,step (a) further comprising apportioning the sterile mononuclear cellsinto a plurality of aliquots, at least one of which is frozen at 86° C.'and stored in the vapor phase of a liquid nitrogen freezer.
 26. Themethod according to claim 7, wherein the myocardial ischemia is atransient ischemia or a chronic myocardial ischemia.
 27. The methodaccording to claim 7, wherein the myocardial ischemia is a peri-infarctborder zone ischemia.
 28. The method according to claim 19, wherein thecytokine is a hematopoietic stem cell mobilizing agent.
 29. The methodaccording to claim 27, wherein the hematopoietic stem cell mobilizingagent is G-CSF, GM-CSF, or a combination thereof.
 30. The methodaccording to claim 19, wherein the vasoactive agent is an angiotensinconverting enzyme inhibitor.
 31. The method according to claim 19,wherein the antiarrhythmic agent is a beta blocker.
 32. The methodaccording to claim 1, wherein the subject is a revascularized subject.33. The method according to claim 14, wherein worsening of congestiveheart failure, worsening of significant arrhythmias, worsening of acutecoronary syndrome, or a combination thereof would lead to prematuredeath in the absence of treatment.